Human anti-IFN-α antibodies

ABSTRACT

Provided are novel IFB-a binding molecules of human origin, particularly human-derived anti-IFN-α antibodies as well as IFN-α binding fragments, derivatives and variants thereof. In addition, pharmaceutical compositions, kits, and methods for use in diagnosis and therapy are described.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/902,409, filed Dec. 31, 2015, now U.S. Pat. No. 10,112,995, issued onOct. 30, 2018, which is a U.S. national stage application under 35U.S.C. 371 and claims the benefit of PCT Application No.PCT/EP2014/064167 having an international filing date of Jul. 3, 2014,which designated the United. States, which PCT application claimed thebenefit of European Patent Application No. 13174995.4 filed Jul. 3,2013, the disclosures of each of which are incorporated herein byreference.

Reference to Sequence Listing

This application contains a Sequence Listing submitted as an electronictext file named “W02015001013SequenceListing.txt”, having a size inbytes of 162 KB, and created on Jul. 30, 2014, The sequence listinginformation recorded in computer readable form is identical to thewritten (on paper or compact disc) sequence listing and includes no newmatter. The information contained in this electronic file is hereby,incorporated by reference in its entirety pursuant to 37 CFR §1.52(e)(5).

FIELD OF THE INVENTION

The present invention generally relates to novel molecules binding IFN-αof mammal, preferably human origin, particularly human monoclonalantibodies as well as fragments, derivatives and variants thereof thatrecognize different subtypes of IFN-α. In particular, recombinant humanpatient-derived anti-IFN-α antibodies and methods of making the same areprovided. In addition, compositions comprising such binding molecules,antibodies and mimics thereof useful in the treatment and diagnosis ofdisorders are described. Furthermore, the present invention relates toautoantibodies as agents for use in immunotherapy as well as targets inthe therapeutic intervention of autoimmune and autoinflammatorydisorders as well as malignancies, such as systemic lupus erythematosus(SLE) and type 1 diabetes mellitus (TIDM). More specifically, thepresent invention relates to monoclonal autoantibodies which have beenisolated from B cells derived from subjects affected with an impairedcentral and/or peripheral tolerance or loss of self-tolerance typicallydue to a mutation in a gene involved in immune regulation.

BACKGROUND OF THE INVENTION

Inappropriate responses of the immune system may cause stressfulsymptoms to the involved organism. Exaggerated immune answers to foreignsubstances or physical states which usually do not have a significanteffect on the health of an animal or human may lead to allergies withsymptoms ranging from mild reactions, such as skin irritations tolife-threatening situations such as an anaphylactic shock or varioustypes of vasculitis. Immune answers to endogenous antigens may causeautoimmune disorders such as systemic lupus erythematosus (SLE), typefor insulin dependent diabetes mellitus (TIDM or IDDM) and differentforms of arthritis.

Immune responses occur in a coordinated manner, involving several cellsand requmng communication by signaling molecules such as cytokinesbetween the cells involved. This communication may be influenced orinhibited by, e.g., interception of the signals or block of therespective receptors.

Cytokines are secreted soluble proteins, peptides and glycoproteinsacting as humoral regulators at nano- to picomolar concentrationsbehaving like classical hormones in that they act at a systemic leveland which, either under normal or pathological conditions, modulate thefunctional activities of individual cells and tissues. Cytokines differfrom hormones in that they are not produced by specialized cellsorganized in specialized glands, i.e. there is not a single organ orcellular source for these mediators as they are expressed by virtuallyall cells involved in innate and adaptive immunity such as epithelialcells, macrophages, dendritic cells (DC), natural killer (NK) cells andespecially by T cells, prominent among which are T helper (Th)lymphocytes.

Depending on their respective functions, cytokines may be classifiedinto three functional categories: regulating innate immune responses,regulating adaptive immune responses and stimulating hematopoiesis. Dueto their pleiotropic activities within said three categories, e.g.,concerning cell activation, proliferation, differentiation, recruitment,or other physiological responses, e.g., secretion of proteinscharacteristic for inflammation by target cells, disturbances of thecell signaling mediated by aberrantly regulated cytokine production havebeen found as a cause of many disorders associated with defective immuneresponse, for example, inflammation and cancer.

Interferons (IFN), consisting from three known protein families, type I,II and III interferons constitute one of the most important classes ofcytokines. All human type I interferons bind to a cell surface receptor(IFN alpha receptor, IFN-αR) consisting of two transmembrane proteins,IFN-αR-1 and IFN-αR-2 leading to JAK-STAT activation, the formation ofISGF3 and subsequent onset of gene expression (Platanias and Fish, Exp.Hematol. (1999), 1583-1592). The composition, receptors and signalingpathways of type I IFNs have been reviewed, e.g., in Stark et al., Annu.Rev. Biochem. (1998), 227-64; Pestka S., Biopolymers (2000), 254-87.Type I interferons build a structurally related family (IFN-α (alpha),IFN-(beta), IFN-K (kappa), IFN-8 (delta), IFN-£ (epsilon), IFN-T (tau),IFN-ω (omega), and IFN-s (zeta)), of which IFN-8 and IFN-T do not occurin humans. Human type I interferon (IFN) genes are clustered on humanchromosome 9p21 and the mouse genes are located in the region ofconserved synteny on mouse chromosome 4. So far, 14 IFN-α genes and 3pseudogenes have been identified in the mouse. In humans 13 IFN-α (orIFN-α) genes (IFN-α1, IFN-α2, IFN-α4, IFN-α5, IFN-α6, IFN-α7, IFN-α8,IFN-α10, IFN-α13, IFN-α14, IFN-α16, IFN-α17 and IFN-α21) and 1pseudogene have been identified, wherein two human IFN-α genes(IFN-α1/IFN-α1 and IFN-α13/IFN-α13) encode for identical proteins (vanPesch et al., J Viral. (2004), 8219-8228).

IFN-y is the sole Type II interferon. It is mainly involved in theinduction of antimicrobial and antitumor mechanisms by macrophagestimulation. The IFN-γ receptor (IFNGR) is a heterodimeric receptorcomprised of two ligand-binding IFNGR1 chains associated with twosignal-transducing IFNGR2 chains (Schroder et al., J. Leukoc. Biol. 75(2004), 163-189; Bach et al., Annu. Rev. Immunol. 15 (1997), 563-591).Type III interferons consist of three subtypes and are also termed IFNλ(IFNλ1 or IL-29, IFNλ2 or IL-28A and IFNλ3 or IL-28B) and haveantiviral, antitumor, and immunoregulatory activity. The IFN-λ receptoris also a heterodimeric complex consisting of a unique ligand-bindingchain, IFN-λR1 (also designated IL-28Rα), and an accessory chainIL-10R2, which is shared with receptors for IL-10-related cytokines (Liet al., J. Leukoc. Biol. 86 (2009), 23-32).

Type I interferons are pleiotropic cytokines with antiviral, antitumorand immunoregulatory functions. Depending on context, they can beanti-inflammatory and tissue protective or proinflammatory and promoteautoimmunity. IFN-1a or 1b is used for the treatment of multiplesclerosis and IFN-α2b therapy for many cancers (melanoma, hemat.malig.). Elevated IFN-α activity has been frequently detected in thesera of patients with systemic lupus erythematosus (SLE) indicating thatIFN-α plays a central role in SLE development (R6nnblom and Alm, J Exp.Med. (2001), F59-F63; CrowMK, Arthritis Rheum. (2003), 2396-2401; Crow MK., Curr Top Microbial. Immunol. (2007), 359-386; Crow M K. Rheum DisClin North Am. (2010), 173-186).

On the other side, a specific expression pattern of interferon-dependentgenes (termed the “interferon signature”) is displayed in the leukocytesof patients with various autoimmune disorders such as SLE, TIDM,Sj6gren's syndrome, Dermatomyositis, Multiple Sclerosis (MS), Psoriasisand rheumatic arthritis (RA) patients. In addition, development ofinflammatory arthritis, MS and TIDM has been repeatedly observed duringIFN-α therapy indicating that IFN-α at least promotes those diseases(Crow M K., Arthritis Res Ther. (2010), Suppl 1:S5). Further datasuggest an involvement of IFN-α in myositis, systemic scleroderma,chronic psoriasis (Higgs et al., Eur Muse Rev (2012), 22-28; Bissonnetteet al., J Am Acad Dermatol (2009), 427-436; Greenberg S A, Arth Res Ther(2010): S4;) and autoimmune thyroiditis (Prummel and Laurberg, Thyroid(2003), 547-551).

Accordingly, wherein depending on the context situation, treatment withType I interferons, as in RA, MS and different leukemia or treatmentwith antibodies neutralizing Type I interferons, e.g., in SLE may beindicated, the very same treatment may be detrimental to the patient bypromoting autoimmunity, inflammation and interferon-treatment relatedtoxicities or even leading to the development of diseases such as MS andTIDM. One factor in these different effects may arise from the factthat, despite that different IFN subtypes activate the same cell surfacereceptor complex, they mediate variable responses which are also celltype dependent (van Pesch et al., J Viral 78 (2004), 8219-8228;Antonelli G., New Microbial. 31 (2008), 305-318; Gibbert et al., PLoSPathog. 8 (2012), e1002868). The treatment therefore, should preferablybe performed in a selective manner, wherein only particular IFN-αsubtypes are administered to a patient, or particular IFN-α subtypes areneutralized, which are associated with a given pathological condition.Regarding IFN-α treatment such selectivity may be obtained by usage ofhighly purified IFN-α preparations for therapeutic purposes (AntonelliG., New Microbiol. 31 (2008), 305-318). However, regarding the use ofIFN-α antibodies it is more difficult to obtain selectivity in respectof specific IFN-α subtypes, because there is a high degree of homologyat the amino acid level with 80-95% homology between the IFN-α subtypesand 50% homology with IFN-β. It would be desirable therefore to providea pool of IFN-α antibodies tolerable in humans of varying specificitytowards all, selected or particular human IFN-α subtypes, which might beselectively used depending on the therapeutic and/or diagnosticindication.

First attempts to achieve this aim have already been made. For example,patent application US 2009/0214565 A1 describes isolation of severalmouse anti-human IFN-α antibodies which neutralize between three andthirteen different subtypes of human IFN-α and U.S. Pat. No. 7,087,726B2 describes a murine anti-human IFN-α antibody and its humanizedversion which recognize seven subtypes of human IFN-α. However, most ifnot all anti-IFN-antibodies provided so far are of murine origin andthus prone to an adverse reaction in humans.

Due to immunological responses to foreign antibodies, as mouseantibodies in humans (RAMA-response; Schroff et al., Cancer Res. 45(1985), 879-885; Shawler et al., J Immunol. 135 (1985), 1530-1535),mostly humanized versions of antibodies are used in present therapeuticapproaches (Chan and Carter, Nature Reviews Immunology 10 (2010),301-316; Nelson et al., Nature Reviews Drug Discovery 9 (2010),767-774). One approach to gain such antibodies was to transplant thecomplementarity determining regions (CDR) into a completely humanframework, a process known as antibody humanization (Jones et al.,Nature 321 (1986), 522-525). This approach is often complicated by thefact that mouse CDR do not easily transfer to a human variable domainframework, resulting in lower affinity of the humanized antibody overtheir parental murine antibody. Therefore, additional and elaboratemutagenesis experiments are often required, to increase the affinity ofthe so engineered antibodies. Another approach for achieving humanizedantibodies is to immunize mice which have had their innate antibodygenes replaced with human antibody genes and to isolate the antibodiesproduced by these animals. However, this method still requiresimmunization with an antigen, which is not possible with all antigensbecause of the toxicity of some of them. Furthermore, this method islimited to the production of transgenic mice of a specific strain.

Another method is to use libraries of human antibodies, such as phagedisplay, as described, for example, for the generation of IL-13 specificantibodies in international application WO 2005/007699. Here,bacteriophages are engineered to display human scFv/Fab fragments ontheir surface by inserting a human antibody gene into the phagepopulation. Unfortunately, there is a number of disadvantages of thismethod as well, including size limitation of the protein sequence forpolyvalent display, the requirement of secretion of the proteins, i.e.antibody scFv/Fab fragments, from bacteria, the size limits of thelibrary, limited number of possible antibodies produced and tested, areduced proportion of antibodies with somatic hypermutations produced bynatural immunization and that all phage-encoded proteins are fusionproteins, which may limit the activity or accessibility for the bindingof some proteins. Similarly, European patent application EP 0 616 640 A1describes the production of auto-antibodies from antibody segmentrepertoires displayed on phage. Phage libraries are generated fromunimmunized humans in this respect (see, e.g., Example 1; page 16, lines43-51; Example 2, at page 17, paragraph [0158], lines 57-58). However,also the methods described in this patent application suffer from abovementioned general disadvantages of antibodies generated from phagelibraries, in comparison to antibodies produced and matured in amammalian, i.e. human body.

The same applies to the most prominent anti-IFNa monoclonal antibodySifalimumab (formerly, MEDI-545) that binds to and specificallyneutralizes most IFN-α subtypes, preventing signaling through the type IIFN receptor. Sifalimumab is said to be a “human” anti-IFNa monoclonalantibody but actually has been derived from humanized mice, i.e. fromthe former company Medarex' UltiMab platform which is based ontransgenic mice in which the largest fraction of the human germlinerepertoire was introduced. Nevertheless, though the amino acid sequencesof the antibodies derived from humanized mice are of human origin theseantibodies are artificial and not truly human as they have not undergoneimmunization, recombination, selection and affinity maturation in ahuman being for which reason there is still the risk of their beingimmunogenic and less effective, in particular compared to human-derivedantibodies.

In view of the above, there is still a need for additional and newcompounds like binding molecules of high specificity for particularhuman IFN-α subtypes, specific for a selected range or for all IFN-αsubtypes which are tolerable in humans either for monotherapy orcombinatorial approaches.

The solution to this problem is provided by the embodiments of thepresent invention as characterized in the claims and disclosed in thedescription and illustrated in the Examples and Figures further below.

SUMMARY OF THE INVENTION

The present invention relates to IFN-α specific human monoclonalantibodies and IFN-α binding fragments thereof. In particular, humanmonoclonal anti-IFN-α antibodies are provided with a selective bindingprofile towards IFN-α subtypes and displaying binding and neutralizingactivity as shown in the Examples and the Figures. Due to theirneutralizing property, the antibodies of the present invention havetherapeutic, prognostic and diagnostic utility, which make them inparticular valuable for applications in relationship with diverseautoimmune or autoinflammatory disorders and conditions associatedwith/involving IFN-α activity in initiation and/or maintenance ofundesired immune responses, such as systemic lupus erythematosus (SLE),various forms of arthritis including but not limited to rheumatoidarthritis (RA), type 1 or insulin dependent diabetes mellitus (TIDM orIDDM), Sj6gren's syndrome, Dermatomyositis, Multiple Sclerosis (MS),psoriasis, chronic psoriasis, myositis, systemic scleroderma, autoimmunethyroiditis and cancer including leukemia (ALL; Einav et al., Oncogene24 (2005), 6367-6375); see as well the section “Background of theinvention” above, describing the IFN-α subtypes and their possibleinvolvement disorders and implications towards possible indications ofthe antibodies of the present invention in related therapeutic,diagnostic and/or prognostic applications.

The antibodies of the present invention are preferably isolated frommammals, in particular humans, which are affected with an impairedcentral and/or peripheral tolerance or loss of self-tolerance which maybe due to or associated with a disrupted or deregulated genesis ofself-tolerance, preferably caused by a monogenic autoimmune disorder.Examples of mammals which provide a particularly suitable source forautoantibodies in accordance with the present invention are mammals,e.g., humans having a disorder associated with a mutation in the AIRE(Autoimmune Regulator) gene such as Autoimmune polyendocrinopathysyndrome type 1 (APS1) (Peterson et al., Nat. Rev. Immunol. 8 (2008),948-957), Autoimmune polyendocrinopathy syndrome type 2 (APS2) (Baker etal., J Clin. Endocrinol. Metab. 95 (2010), E263-E270) andimmunodysregulation polyendocrinopathy enteropathy X-linked syndrome(IPEX) (Powell et al., J Pediatr. 100 (1982), 731-737; Ochs et al.,Immunol. Rev. 203 (2005), 156-164). Preferably, the patients from whomthe antibodies were isolated are APS1 patients characterized by beingsymptom-free for Lupus erythematodes (SLE) and displaying seroreactivityagainst dsDNA and at least one at least one of the human IFN-α subtypes.

In particular, in accordance with the present invention for the firsttime human and human patient derived anti-IFN-α antibodies are providedwith different IFN-α binding profiles and IFN-α neutralizing activity,thereby alone or in combination covering substantially all IFN-αsubtypes.

Therefore, in one aspect the present invention generally relates to highaffinity neutralizing monoclonal antibodies to several IFN-α subtypes.In a further aspect, the present invention relates to human monoclonalantibodies (mAbs, or MABs) against several IFN-α subtypes, i.e. at leastone of the human IFN-α subtypes IFN-α1/13 (1/13 IFN-α1 b), IFN-α2,IFN-α4, IFN-α5, IFN-α6, IFN-α8, IFN-α10, IFN-α14 or IFN-α21, describedin detail below, which are considered to be safe and effectivetherapeutics for disorders in which those cytokines are involved. In oneembodiment, the IFN-α binding molecule of the present invention does notbind and/or neutralize to any significant extent IFN-(beta interferon,IFNB), IFN-γ (gamma interferon, IFNG) or IFN-ω (interferon omega,IFN-ω).

In another embodiment, in accordance with the present inventionanti-IFN-α antibodies are provided, which in addition bind to andneutralize the activity of IFN-ω, respectively, hitherto described to beunrelated to IFN-α in its antigenic properties, as it usually does notcross-react with antisera or monoclonal antibodies in immunoassays orantiviral neutralization bioassays. Thus, in one aspect the presentinvention relates to novel IFN binding molecules, preferablyhuman-derived monoclonal antibodies as well as fragments andbiotechnological derivatives thereof which are capable of binding to/andor neutralizing the activity of at least one human IFN-α subtype and ofhuman IFN-ω. Preferably, the binding and neutralizing activity,respectively, of the IFN-binding molecule is essentially the same or atleast in the same order of magnitude for IFN-α subtype(s) and IFN-ω.

Naturally, the present invention extends to nucleic acids, in particularcDNA encoding at least one variable, constant and/or complementaritydetermining region of the antibodies of the present invention, vectorscomprising such nucleic acids, antibody producing cell lines andrecombinant cells. The present invention further relates topharmaceutical compositions, diagnostic assays and kits that comprisethe binding molecules or peptides recognized by the antibodies isolatedin accordance with the present invention and to therapeutic methodsbased thereon.

In addition, the present invention relates to a process for themanufacture of a human-derived anti-IFN-α and anti-IFN-α/IFN-ωmonoclonal antibody, respectively, or an IFN-binding fragment orbiotechnological derivative thereof or of a composition comprising theanti-IFN-α and anti-IFN-α/IFN-ω monoclonal antibody, respectively, or anIFN-binding fragment or biotechnological derivative thereof, whichmanufacture comprises the step of preparation of the antibody,IFN-binding fragment or biotechnological derivative thereof byexpression in a recombinant host organism of transforming DNA encodingthe antibody, IFN-binding fragment or biotechnological derivativethereof. In one embodiment, the composition is a pharmaceuticalcomposition, wherein the step of preparation of the antibody,IFN-binding fragment or biotechnological derivative thereof is followedby admixing the antibody, IFN-binding fragment or biotechnologicalderivative thereof with a pharmaceutically acceptable carrier in themanufacture of a pharmaceutical composition.

While the invention is illustrated and described by way of reference tothe human-derived antibody originally obtained in the experimentsperformed in accordance with the present invention and described in theExamples it is to be understood that the antibody or antibody fragmentof the present invention include synthetic and biotechnologicalderivatives of an antibody which means any engineered antibody orantibody-like IFN binding molecule, synthesized by chemical orrecombinant techniques, which retains one or more of the functionalproperties of the subject antibody, in particular its neutralizingactivity towards IFN-α and IFN-ω. Thus, while the present invention maybe described for the sake of conciseness by way of reference to anantibody, unless stated otherwise synthetic and biotechnologicalderivatives thereof as well as equivalent IFN binding molecules aremeant and included with the meaning of the term antibody.

Further embodiments of the present invention will be apparent from thedescription and examples that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Amino acid sequences of the variable region, i.e. heavy chainand kappa/lambda light chain (VH, VL) of anti-IFN-α specific humanantibodies of the present invention. IgG1, kappa, anti-IFN-α specificantibodies A: 5D1; B: 13B11; C: 19D11; D: 25C3; E: 26B9; F: 31B4, G:8H1, H: 12H5 and I: 50E11. Framework (FR) and complementaritydetermining regions (CDRs) are indicated with the CDRs being underlined.Italic amino acids indicate sequences which have not been sequenced butobtained from database. Due to the cloning strategy the amino acidsequence at the N-terminus of the heavy chain and light chain maypotentially contain primer-induced alterations in FR1, which however donot substantially affect the biological activity of the antibody.

FIG. 2: Cross-competition—Epitope mapping. Differential binding ofexemplary anti-IFN-α MABs of the present invention to distinct bindingsites was investigated in cross-competition experiments on A: IFN-α2, B:IFN-α4 and C: IFN-α14. As an IFN-α non-binding control (hlgG1) a humanantibody binding to an unrelated antigen has been used.

FIG. 3: EC50 ELISA determination. EC50 binding of hMABs 5D1, 13B11, 25C3and 26B9 to A: IFN-α2, B: IFN-α4 and C: IFN-α14. EC50 binding toIFN-α2/-4/-14 of hMABs D: 19D11 and E: 31B4.

FIG. 4: Correlating the presence of known disease-associated andprotective autoantibodies in APS1 patients indicates that IFN-αautoantibodies prevent the onset of lupus erythematosus in APS1 patients(columns dsDNA to IFNα14). Target: Coding number of the particularAPS1-patient examined in the correlation study. Anti-dsDNA antibodiesare highly specific for SLE and used in the diagnosis of the disease. NoAPS1 patients have lupus despite the frequent presence of anti-dsDNAantibodies as assessed by ProtoArray analysis (Life Technologies). APS1patients display pronounced seroreactivity against several IFN-αsubtypes which are clinically-relevant drug targets involved in manylupus-implicated molecular mechanisms. Circles indicate the presence,empty cells the absence of particular antibodies. Patients 2, 4, 13 and21 (black arrows) are T1DMs. Patients 10, 14, 16, 17 and 18 (whitearrows) have characteristics of being T1DM but are not. The resultssuggest differential neutralizing activity in serum of APS1 patientssuffering from T1DM and these who are not. As can be inferred from FIGS.31 and 32 this difference observed here seems to be due to differencesin titers of anti-IFN antibodies in both patient classes, with muchhigher antibody titers in APS1 patients not suffering from T1DM, ratherthan differences in the neutralizing activity of individual anti-IFNantibodies.

FIG. 5: Human-derived anti-IFN-α monoclonal antibodies neutralizerhIFN-α-mediated STAT1 activation in HEK 293T cells. HEK 293T cells wereeither left untreated (−) or stimulated (+) with recombinant humanrhIFN-αs or IFN-Gaussia luciferase fusion proteins (g1 IFNs), in theabsence of antibodies or in the presence of human-derived IFN-αmonoclonal antibodies or a human control IgG as indicated (Ctrl). Celllysates were subjected to SDS-PAGE and phosphorylated STAT1 levels(pSTAT1) were visualized in Western blots. Total STAT1 levels or tubulinlevels serve as loading control. Antibody concentration: 5 μg/ml.rhIFN-α concentration: 10 ng/ml (rhIFN-α1), 2 ng/ml (all other rhIFNs);g1 IFN-α-containing supernatants of 293T cells transiently expressingIFN-Gaussia luciferase fusion proteins were used at their respectiveEC80 dilutions. A1: Control: detection of total and phosphorylated STAT1(pSTAT1) by Western Blots (WB) in 293T cells after 100 ng/mlIFN-α2/-4/-14 stimulation. 293T-cells stimulation by IFN-α2/-4/-14. (i):phosphorylated STAT1, (ii): total STAT1, (iii): Tubulin loading control.Time: indicates treatment duration of the cells with the respective IFN.After stimulation phosphorylated STAT 1 can be observed for all threeIFN-α subtypes. Positions of respective molecular weight (kDa) standardbands are indicated on the left of the blot for comparison. A2: Control:detection of total and phosphorylated STAT1 (pSTAT1) by Western Blots(WB) in 293T cells after stimulation with different doses of rhIFN-α1,rhIFN-α2 and rhIFN-α16. B: IFN-α1, IFN-α2, IFN-α4, g1 IFN-α5 and IFN-α6stimulation. All exemplary antibodies except 5D1 neutralize IFN-α1efficiently, and all exemplary antibodies neutralize IFN-α2, IFN-α4 andg1 IFN-α5. Exemplary antibodies 25C3, 5D1 and 13B11 display weakerneutralization of IFN-α6. C: IFN-α7, g1 IFN-α8, IFN-α10, IFN-α14 andIFN-α16 stimulation. All exemplary antibodies neutralize IFN-α7. Allexemplary antibodies except 25C3 and 13B11 neutralize g1 IFN-α8efficiently. IFN-α10 is efficiently neutralized by all exemplaryantibodies except 25C3, while IFN-α16 is efficiently neutralized only byexemplary antibodies 19D11, 5D1 and 13B11. D: IFN-α17, IFN-α21, IFN-ω,IFNB and g1 IFNG stimulation. All exemplary antibodies neutralizeIFN-α17, and all exemplary antibodies except 13B11 efficientlyneutralize IFN-α21. IFN-ω is apparently only efficiently neutralized byexemplary antibodies 26B9 and 31B4. None of the exemplary antibodiesneutralizes IFNB or g1 IFNG.

FIG. 6: Schematic representation of the Firefly-Luciferase reporterconstruct and the Renilla-Luciferase construct used as internalnormalization control. TRETranscription response element;CMV—cytomegalovirus.

FIG. 7: Human-derived anti-IFN-α monoclonal antibodies neutralizerhIFN-α-induced ISRE-Luciferase reporter gene activation in HEK 293Tcells. A and B: Testing of exemplary anti-IFN-α antibodies 19D11, 25C3,26B9 and 31B4. HEK 293T cells transiently expressing ISRE dualluciferase reporter constructs were either left untreated (−) orstimulated with rhIFNs (+), in the absence of antibodies or in thepresence of human-derived IFN-α monoclonal antibodies or a human controlIgG as indicated. rhIFN concentration: 2 ng/ml; antibody concentration:5 μg/ml, C and D: Testing of exemplary anti-IFN-α antibodies 5D1 and13B11. HEK 293T cells transiently expressing ISRE dual luciferasereporter constructs were treated as in (A/B) and ISRE reporter activitywas analyzed after 24 hours. rhIFN-α concentration: 1 ng/ml; antibodyconcentration: 5 μg/ml.) A/C: measurements of the relative luciferaseunits, B/D: calculation of the expressional fold change. E:Neutralization of rhIFN-α1, A2, A4, A5, A6, A7, A8, A10, A14, A16, A17,A21, rhIFN-ω and rhIFNB by exemplary human-derived monoclonal antibodiesof the present invention 8H1 and 12H5. Exemplary antibody 8H1 fullyneutralizes IFN-ω, together with IFN-α1, A4, A5, A6, A7, A10, A16, A17and A21, while displaying slightly weaker neutralization of IFN-α2, A8and A14. Exemplary antibody 12H5 neutralizes all IFN-α subtypes and notIFN-ω. Neither exemplary antibody 8H1 nor 12H5 neutralizes IFNB. HEK293T MSR were treated as in A and ISRE reporter activity was analyzedafter 24 hours. rhIFN concentration: 10 ng/ml (IFN-α1), 1.3 ng/ml(IFN-α16), 4 ng/ml (IFN-α21), 1 ng/ml (IFNB) and 2 ng/ml (all otherIFNs). Antibody concentration: 5 μg/ml.

FIG. 8: IC 50 analysis of exemplary human-derived IFN-α mAb 26B9 byISRE-Luciferase reporter neutralization assay. IC 50 neutralizationgraphs by exemplary antibody 26B9 of A: IFN-α2; B: IFN-α4; C: IFN-α5; D:IFN-α8; E: IFN-α14. IC 50 analysis results of a confirmative round ofneutralization assays for the exemplary antibody 26B9 are shown of F:IFN-α1, G: IFN-α2, H: IFN-α4, I: IFN-α5, J: IFN-α6, K: IFN-α7, L:IFN-α8, M: IFN-α10, N: IFN-α14, O: IFN-α16, P: IFN-α17, Q: IFN-α21 andR: IFN-ω. IC 50 data is summarized in Table 4. RLU at theY-axis=relative light units, as in foregoing figures.

FIG. 9: IC50 analysis of exemplary human-derived anti-IFN-α mAb 25C3 byISRE Luciferase reporter neutralization assay. Assay performed asdescribed in FIG. 8. IC50 neutralization graphs by exemplary antibody26B9 of: A: IFN-α2; B: IFN-α4; C: IFN-α5; D: IFN-α8 and E: IFN-α14.

FIG. 10: IC50 analysis of exemplary human-derived anti-IFN-α mAb 19D11by ISRE Luciferase reporter neutralization assay. Assay performed asdescribed in FIG. 8. IC50 graphs of neutralization by exemplary antibody19D11 of: A: IFN-α2; B: IFN-α4; C: IFN-α5; D: IFN-α8; E: IFN-α14. IC 50neutralization graphs of a confirmative experimental round by exemplaryantibody 19D11 of F: IFN-α1, G: IFN-α2, H: IFN-α4, I: IFN-α5, J: IFN-α6,K: IFN-α7, L: IFN-α8, M: IFN-α10, N: IFN-α14, O: IFN-α16, P: IFN-α17 andQ: IFN-α21. IC 50 data is summarized in Table 4.

FIG. 11: A: Determination and comparison of binding of exemplary MABs19D11, 25C3, 26B9, 5D1 and 13B11 of the present invention to IFN-α1 andIFN-α2 (ImmunoTools) by ELISA. Exemplary anti-IFN-α-α antibody 5D1 doesnot cross-react with IFN-α1. B: Determination and comparison of bindingof exemplary MABs 5D1, 13B11, 19D11, 25C3, 26B9 and 31B4 of the presentinvention to IFN-α8 and IFN-α14 (IFN-Gaussia luciferase fusion proteins)by LIPS. Exemplary MAB 13B11 does not have cross-reactivity with IFN-α8(g1IFN-α8). C: Determination and comparison of binding of exemplary MABs5D1, 13B11 19D11, 25C3, 26B9 and 31B4 of the present invention toIFN-α5, IFN-α6, IFN-α8, IFN-α21 (all from PBL) and IFN-α14 (ATGen).Exemplary anti-IFN-α-α antibody 13B1 does not cross-react with IFN-α8and IFN-α21. Antibody 19D11 cross-reacts with a lower affinity withIFN-α21 than with the other IFN-α subtypes. MABs were tested at 1 μg/mlin (C).

FIG. 12: LIPS assay—determination of binding of the antibodies of thepresent invention to different IFN-α subtypes (IFN-Gaussia luciferasefusion proteins). Binding of the exemplary antibodies of the presentinvention to A: IFN-α5; B: IFN-α6 and C: INFA8. Exemplary anti-IFN-αantibody 13B11 shows no substantial cross-reactivity with IFN-α8. As anIFN-α non-binding control (hlgG1) an human antibody binding to anunrelated antigen was used.

FIG. 13: LIPS assay—determination of binding characteristics of theexemplary antibodies 5D1, 13B11, 19D11, 25C3, 26B9 and 31B4 of thepresent invention towards IFN-α1, IFN-α2, IFN-α5, IFN-α6, IFN-α8,IFN-α10, IFN-α14, IFN-α16 and IFN-α21 (IFN-Gaussia luciferase fusionproteins). As an IFN-α non-binding control (hIgG1) a human antibodybinding to an unrelated antigen was used. All antibodies were tested at0.5 μg/ml. All IFN-α subtypes used in FIGS. 12 and 13 are IFN-α-Gaussialuciferase fusion proteins (g1IFN-αs).

FIG. 14: Ear inflammation assay-test of the proinflammatory effect ofhuman IFN subtypes in mice. A: Exemplary 6-day experimental timeline. B:CytoEar ear thickness measurements calculated as fold change relative today 0 measurements than normalized to relevant PBS controls, for eachcohort. B1: Overview of the effect of all normalized measurements. B2:Effects of the IFNa2a and IFNa2b injections. B3: Effects of the IFNa4and IFNa14 injections. All four human IFN-α subtypes tested were able tosignificantly induce ear swelling following ID. All ears were markedlythicker than PBS treated ears. IFNa14 was the most potentproinflammatory agent. Mean +/−SEM, 11-3 or ID=intradermal cytokineinjections, M=Measurements—ear thickness and animal weight, S=Sacrificeof the animals; short arrows—cytokine injections; long arrows—exemplarydays of anti-IFN-α antibody injections.

FIG. 15: Ear inflammation assay—test of the proinflammatory effect ofhuman IFN subtypes in mice. CytoEar ear thickness measurements are shownas absolute values (mm) for each cohort. A: Overview of the effect ofall measurements. B: Effects of the IFNa2a and IFNa2b injections. C:Effects of the IFNa4 and IFNa14 injections. All indications as in FIG.14.

FIG. 16: Ear inflammation assay—test of the proinflammatory effect ofhuman IFN subtypes in mice. CytoEar ear thickness measurements are shownfold change from Day 0 for each cohort. The thickness on Day 0 has beenset as 1. A: Overview of the effect of all measurements. B: Effects ofthe IFNa2a and IFNa2b injections. C: Effects of the IFNa4 and IFNa14injections. All indications as in FIG. 14.

FIG. 17: Summary of the ear inflammation assay in respect of human IFN-αsubtype capabilities for induction of ear inflammation. P valuesobtained by 2-way ANOVA testing, ns (not significant)=P>0.05; *=P≤0.05;**=P≤0.01; ***=P<0.001, ****=p<0.0001. All ears were markedly thickerthan PBS treated ears; this was significant in all groups after the 2ndID, from Day 3 until the end of the experiment.

FIG. 18: LIPS assay—determination and comparison of bindingcharacteristics of the exemplary anti-IFN-α antibodies 5D1, 13B11,19D11, 25C3, 26B9 and 31B4 of the present invention towards IFNγ (IFNG),IFN-β1(IFNB1), IFNε (IFNE), IFN-ω (IFN-ω), three IFNλ (IL28A, IL28B andIL29) and IFN-α10 (all IFN-Gaussia luciferase fusion proteins). A:Binding of the antibodies towards the different IFN-types. B: Reciprocaldiagram, binding of the IFNs by the antibodies. Exemplary antibodies26B9 and 31B4 show binding of IFN-ω (IFN-ω) besides their affinitytowards IFN-α10. No substantial binding of the exemplary antibodies ofIFNβ1(IFNB1). IFNε (IFNE), the three IFNλ and IFNγ (IFNG) could beobserved.

FIG. 19: Cross-reactivity of MABs towards mouse IFN-αs (ELISA).hIgG1=antibody of non-IFN-α related specificity (negative control). A:Test of cross-reactivity of the exemplary antibodies 5D1, 13B11, 19D11,25C3, 26B9 and 31B4 against human and murine IFN-α2, IFN-α4 and IFN-α14in an ELISA assay. Except antibody 25C3 showing cross-reactivity towardsmurine IFN-α2, the other antibodies show no or only residual bindingspecificity towards the murine IFN-α subtypes tested. B:Cross-reactivity of MABs on mouse IFN-αs (LIPS-assay). Antibodies 5D1and 19D11 show cross-reactivity towards murine g1mIFN-α1, wherein theother antibodies only show reactivity below the level observed for thenegative control. None of the antibodies shows cross-reactivity towardsmurine IFN-α subtype g1mIFN-α9.

FIG. 20: IC 50 analysis of exemplary human-derived anti-IFN-α mAb 8H1 byISRE Luciferase reporter neutralization assay. IC 50 neutralizationgraphs of exemplary antibody 8H1. A: IFN-α1, B: IFN-α2, C: IFN-α4, D:IFN-α5, E: IFN-α6, F: IFN-α7, G: IFN-α8, H: IFN-α10, I: IFN-α14, J:IFN-α16, K: IFN-α17. L: IFN-α21 and M: 20 IFN-ω. IC 50 data issummarized in Table 4.

FIG. 21: IC 50 analysis of exemplary human-derived anti-IFN-α mAb 12H5by ISRE Luciferase reporter neutralization assay. IC 50 neutralizationgraphs of exemplary antibody 12H5. A: IFN-α1, B: IFN-α2, C: IFN-α4, D:IFN-α5, E: IFN-α6, F: IFN-α7, 25 G: IFN-α8, H: IFN-α10, I: IFN-α14, J:IFN-α16, K: IFN-α17 and L: IFN-α21. IC 50 data is summarized in Table 4.

FIG. 22: IC 50 analysis of exemplary human-derived anti-IFN-α mAb 50E111 by ISRE Luciferase reporter neutralization assay. IC 50 neutralizationgraphs of exemplary antibody 50E11. A: IFN-α1, B: IFN-α2, C: IFN-α4, D:IFN-α5, E: IFN-α6, F: IFN-α7, G: IFN-α8. H: IFN-α10, I: IFN-α14, J:IFN-α16, K: IFN-α17, L: IFN-α21 and M: IFN-ω. IC 50 data is summarizedin Table 4.

FIG. 23: Human-derived anti-IFN-α monoclonal antibodies neutralizebinding of IFN-Gaussia luciferase fusion proteins to HEK 293T MSR cellsendogenously expressing IFN receptors in the luminescent cellularbinding assay. Cells were incubated with supernatants of HEK 293T cellsexpressing Interferon-Gaussia luciferase fusion proteins in the absenceof inhibitors (−) or in the presence of competitive inhibitors asindicated. A: Schematic representation of the luciferase-basedchemiluminescent cellular binding assay. A ligand of interest (3) isfused to Gaussia luciferase (4). The fusion protein is bound to cells(1) expressing receptors for the ligand of interest (2). After removalof unbound fusion proteins a luciferase substrate is added (5) and lightemission is recorded (6). The light output is proportional to the amountof bound fusion protein. Anti-ligand antibodies that compete with thereceptors for binding of the ligand of interest (7) lead to a decreasein bound ligand and reduced light output. B: Control: HumanIFN-α-Gaussia luciferase fusion proteins bind specifically to HEK 293TMSR cells. Binding of IFN-α5-Gaussia luciferase fusion proteins (g1IFN-α5) to HEK 293T MSR cells is inhibited by unlabeled rhIFN-α2 (3μg/ml) and by exemplary human-derived monoclonal IFN-α antibody 19D11(1.7 μg/ml). A human control antibody (huIgG, 15 μg/ml) shows no effect.C: Binding of g 1 IFN-α2, A4, A5, A6, A7, A8, A10, A14, A16, A 17 andA21 fusion proteins to HEK 293T MSR cells is inhibited by exemplaryhuman-derived monoclonal IFN antibody 19D11. Binding of g1 IFNB andIFN-ω is unaffected by 19D11. Binding of all g1 IFNs is unaffected by acontrol human antibody (huIgG). Antibody concentration: 5 μg/ml. D:Binding neutralization of g1 IFN-α16 and g1 IFN-ω by exemplaryantibodies 19D11 and 26B9. Cells were treated with g1 IFN-α16 or g1IFN-ω as indicated in the absence of antibodies (−) or in the presenceof exemplary antibodies 19D11, 26B9 or of a control human antibody(huIgG). Antibody concentration: 10 μg/ml. Exemplary antibody 19D11 ismore potent than 26B9 at neutralizing g1 IFN-α16. Exemplar antibody 26B9efficiently blocks binding of g1 IFN-ω to its receptors on 293T MSRcells, while exemplary antibody 19D 11 shows no apparent effect againstthis ligand.

FIG. 24: Human IFN-ω-Gaussia luciferase fusion proteins bindspecifically to HEK 293T MSR cells expressing a transmembrane anti-IFN-ωmAb. HEK 293T MSR cells were reverse-transfected with the indicatedamounts of cDNA encoding a membrane-bound version of anti-IFN-ω mAb 26B9(26B9-TM) or empty vector (Mock). Forty-eight hours after transfection,IFN-ω-Gaussia luciferase was added (g1 IFN-ω) and binding was analysedin the chemiluminescent cellular binding assay. A: Control: 26B9-TM isexpressed at the cell surface of transfected HEK 293T MSR cells. Surfaceantibody expression was analysed 48 hours after transfection in acell-based ELISA. B: g1 IFN-ω specifically binds to cells expressing26B9-TM in the luminescent cellular binding assay.

FIG. 25: Crosscompetition assay of anti-IFN-ω antibodies. Binding of g1IFN-ω to 26B9-TM is competed dose-dependently by soluble 26B9 and by theclonally related 31B4 antibody. In contrast, binding is not affected bya control IgG or by exemplary anti-IFN-ω antibody 8H1.

FIG. 26: SPR analysis. A: Detailed analysis of the sensograms concerningof the binding of human A1/B1: IFN-α2b, A2/B2: IFN-α4, A3/B3: IFN14 andA4/B4: IFNω to exemplary antibodies 19D11 (A1-A4) and 26B9 (B1-B4) ofthe present invention. A 1:1 binding kinetic was observed. The antigenswere injected in concentrations of 1 nM, 2.5 nM, 5 nM, 10 nM, 15 nM, 25nM, 50 nM and 100 nM. Calculated Affinities (KD values [M]) areindicated in the diagrams. C: The plot shows the kinetic parametersderived from the fitted curves for the association (on-rate ka) anddissociation (off-rate kd) of all tested antibodies. Dashed diagonalsindicate affinities (KD). D: KD values of exemplary antibodies of thisinvention in comparison with SPR literature value of Biotin-Straptavidinbinding. The affinities towards human IFN-α4 and IFN-α14 are in thesub-picomolar range and in sub-nanomolar range for IFN2b, respectively.26B9 also binds human IFNω with a sub-picomolar affinity.

FIG. 27: Epitope mapping. A: Binding of antibodies of the presentinvention to full length antigens coupled to the microarray. The Y-axisindicated the fluorescence intensity (RFU) upon detection with aCy5-conjugated secondary antibody. B: Primary peptide array of 18merpeptides of human IFN-α2 against the antibody 19D11 of the presentinvention. In the lower panel the peptides covering the sequence fromAsparagine 65 to Lysine 98 and from Lysine 117 to Serine 150 aredepicted. The antibody 19D11 binds specifically to peptides 19 and 32.C: Primary peptide array of 18mer peptides of human IFN-α2 against theantibody 26B9 of the present invention. In the lower panel the peptidescovering the sequence from Aspartic acid 77 to Lysine 110 are depicted.The antibody 26B9 binds specifically to peptide 22. D: Primary peptidearray of 18mer peptides of human IFN-αW against the antibody 26B9 of thepresent invention. In the lower panel the peptides covering the sequencefrom Methionine 102 to Alanine 135 are depicted. The antibody 26B9 bindsspecifically to peptide 23.

FIG. 28: Ear inflammation assay CytoEar IFN-α14. Testing the effect ofdifferent IFN-α blocking antibodies of the present invention followinghIFN-α14 induced inflammation. To induce inflammation 20 μl IFNa 14 wasinjected per ear at a concentration of 25 μg/ml. Measurement ofthickness was also performed before giving the injection, and with twomeasurements per ear. A: Exemplary 10-day experimental timeline. B:Overview of the experimental treatment of the experimental animal groupsA to I. Ref. A—IFN-α-specific reference antibody. CytoEar ear thicknessmeasurements calculated as fold change relative to day 0 measurementsthan normalized to relevant PBS controls, for each cohort. C: Overviewof the effect of all normalized measurements. D: Effects of 26B9treatment following IFNa14 injections. E: Effects of 19D11 treatmentfollowing IFNa14 injections. F: Effect of the treatment with referenceanti-IFN-α antibody Ref. A following IFNa14 injections. Treatment withantibodies 26B9 and 19D11 (significant reduction of ear thickness atdays 7, 9, 10, respective at days 4, 7-10 for 19D11) of the presentinvention leads to pronounced reduction of the ear thickness resultingfrom IFNa14 injections compared to the control treatment with IgG (ofIFN-α non-related binding specificity) and of treatment with Ref. A.Mean +/−SEM, ID=intradermal cytokine injections, M=Measurements—earthickness, S=Sacrifice of the animals; ID—cytokine injections; testedantibodies 26B9, 19D11, Ref.A and the control IgG were injected at day 0(IP).

FIG. 29: Ear inflammation assay CytoEar IFN-α5. Testing the effect ofdifferent IFN-α blocking antibodies of the present invention followinghIFN-α5 induced inflammation. To induce inflammation 20 μl IFNa5 wasinjected per ear at a concentration of 25 μg/ml. Measurement ofthickness was also performed before giving the injection, and with twomeasurements per ear. A: Exemplary 10-day experimental timeline. B:Overview of the experimental treatment of the experimental animal groupsA to I. Ref. A—reference IFN-α-specific antibody. CytoEar ear thicknessmeasurements calculated as fold change relative to day 0 measurementsthan normalized to relevant PBS controls, for each cohort. C: Overviewof the effect of all normalized measurements. D: Effects of 26B9treatment following IFNa5 injections. E: Effects of 19D11 treatmentfollowing IFNa5 injections. F: Effect of the treatment with referenceanti-IFN-α antibody Ref. A following IFNa5 injections. Treatment withantibodies 26B9 and 19D11 of the present invention leads to a reductionof the ear thickness resulting from IFNa5 injections (significantreduction for 26B9 at days 4, 6, 7, 8 and 9: for 19B11 at days 7-9). Seedescription of FIG. 26 for further details. Tested antibodies 26B9,19D11, Ref A and the control IgG were injected at day 0 (IP).

FIG. 30: Ear inflammation assay CytoEar IFNw. Testing the effect ofdifferent IFN-α blocking antibodies of the present invention followinghIFNw (IFNω) induced inflammation To induce inflammation 20 μl IFNw wasinjected per ear at a concentration of 6.25m/ml, 125 ng/ear. Measurementof thickness was also performed before giving the injection, and withtwo measurements per ear. A: Exemplary 10-day experimental timeline. B:Overview of the experimental treatment of the experimental animal groupsA to I. Ref. A—reference IFN-α specific antibody. CytoEar ear thicknessmeasurements calculated as fold change relative to day 0 measurementsthan normalized to relevant PBS controls, for each cohort. C: Overviewof the effect of all normalized measurements. D: Effects of 26B9treatment following IFNw injections. E: Effects of 19D11 treatmentfollowing IFNw injections. F: Effect of the treatment with referenceanti-IFN-α antibody Ref. A following IFNw injections. Treatment withantibody 26B9 of the present invention leads to a significant reductionof the ear thickness resulting from IFNw injections at experimental Day9. Treatment with 19D11 or Ref.A leads to apparently none or very slightreduction of the ear thickness (not significant (ns) compared to controlIgG injections at all days). See description of FIG. 26 for furtherdetails. Tested antibodies 26B9, 19D11, Ref.A and the control IgG wereinjected at day 0 (IP).

FIG. 31: Comparison of IFN neutralizing activity in serum of APS1/APECED patients with type 1 diabetes (T1D) or without (N). A: IFN-α1,B: IFN-α2a, C: IFN-α4, D: IFN-α5, E: IFN-α6, F: IFN-α7, G: IFN-α16, H:IFN-α17, I: IFN-α21 and J: IFN-ω.

FIG. 32: Comparison of IFN neutralizing activity in serum of APS1/APECEDpatients with type 1 diabetes (T1D) or without (N). As in FIG. 31however with logarithmic scale at the Y-axis for a better visualizationof the neutralizing activity measured in serum of APS 1/APECED patientswith T1D. A: IFN-α1, B: IFN-α2a, C: IFN-α4, D: IFN-α5, E: IFN-α6, F:IFN-α7, G: IFN-α8, H: IFN-α10, I: IFN-α14, J: IFN-α16, K: IFN-α17, andL: IFN-α21. APS1 patients not suffering from T1D (N) show for allantibodies a higher titer in their serum than T1D-APS1-patients. Thistiter difference may indicate differential therapeutic requirements inboth patient groups.

DETAILED DESCRIPTION OF THE INVENTION

The present invention generally relates to novel molecules binding IFN-αof mammal, preferably human origin, particularly patient-derived humanmonoclonal antibodies as well as fragments, derivatives and variantsthereof that recognize and more importantly are capable of neutralizingthe activity of different subtypes of IFN-α; see also the backgroundsection supra for description of the IFN-α subtypes, including IFN-α1,IFN-α2, IFN-α4, IFN-α5, IFN-α6, IFN-α7, IFN-α8, IFN-α10, IFN-α13,IFN-α14, IFN-α16, IFN-α17 and IFN-α21, wherein the IFN-α1 and IFN-α13genes encode for the same IFN-α1/13 subtype.

As described in the Examples, the exemplary antibodies of the presentinvention have been isolated by a method subject of and disclosed inapplicant's international application WO2013/098419 A1, based onscreening the sera of patients with an impaired central and/orperipheral tolerance or loss of self-tolerance, such as APECED/APS1patients for autoantibodies against IFN-α proteins as well as using anovel method of isolating antibodies from B cells of subject sufferingfrom an autoimmune disorder of auto-inflammatory disease, which issubject of and disclosed in applicant's international applicationWO2013/098420 A1.

Experiments performed in accordance with the present invention weredirected towards the provision of IFN-α binding molecules of selectiveIFN-α subtype specificities, i.e. antibodies and IFN-α binding fragmentsthereof which immunoreactivity has been shown in APECED/APS1 patients asprotective against the onset of, e.g., SLE or at least reducingmanifestation of SLE symptoms.

Accordingly, in a first general aspect the present invention provides ahuman-derived anti-interferon-alpha (IFN-α) antibody or an IFN-α bindingfragment, synthetic or biotechnological derivative thereof showing abinding specificity and preferably neutralizing activity towards all oronly a sub-range or particular IFN-α subtypes. In a preferred embodimentof the present invention the anti-IFN-α antibody or IFN-α bindingfragment

(i) binds to human IFN-α subtypes IFN-α2, IFN-α4, IFN-α5, IFN-α6,IFN-α10 and IFN-α14;

(ii) binds to at least one human IFN-α subtype IFN-α1/13 (IFN-α1b),IFN-α8, IFN-α16 and/or IFN-α21; and/or

(iii) is capable of neutralizing a biological activity of at least oneof the human IFN-α subtypes. Accordingly, the present invention providesa pool of antibodies of different IFNa subtype specificity differentfrom and providing a broader application profile than antibodies knownin the art.

All IFNα subtypes utilize the heterodimeric IFNα/receptor (IFN-αR) andgenerate a signal in the receiving cell via cytoplasmic tyrosine kinasesTyk2 and Jak1 performed phosphorylation and concomitant activation ofSTAT1 and STAT2 (signal transducers and activators of transcription).The STAT proteins translocate to the nucleus and activate the expressionof genes that have either GAS or ISRE sites, or both, in their promoters(Borden et al., Nat. Rev. Drug Discov. 6 (2007), 975-990; Hu et al.,Immunol. Rev. 226 (2008), 41-56; van Boxel-Dezaire et al., Immunity 25(2006), 361-372). This activation mechanism has been used within thepresent invention as well as for designing IFN-activity measurement invitro methods, such as the cell based STAT (signal transducers andactivators of transcription) activation assay and the ISRE (Interferonstimulated response element) reporter gene assay as described and usedherein (Cignal Reporter Assay, Qiagen), e.g., in Example 3 and in FIGS.5 to 7 to monitor the neutralizing abilities of the antibodies of thepresent invention. As described in detail therein, the antibodies of thepresent invention have been found to have a potent neutralizing activitytowards several IFN-α subtypes as specified in detail further below.

Therefore, in one embodiment of the present invention, the biologicalactivity neutralized by the antibody or IFN-α binding fragment of thepresent invention is IFN-α signaling in a cell based STAT (signaltransducers and activators of transcription) activation assay, in anISRE (Interferon stimulated response element) reporter gene assay,and/or in an cellular binding assay (see Example 9).

Furthermore, the binding affinities of the antibodies of the presentinvention have been tested by ELISA, LIPS and cellular binding assays asdescribed herein, e.g., in Examples 1, 2, 6 and 9 and shown in FIGS. 3,7 to 13, and 18 to 27. In accordance with the results of theseexperiments, the present invention provides several exemplary anti-IFN-αantibodies showing differential binding affinities towards distinctIFN-α subtypes, which exemplify the binding and neutralizationcharacteristics of the IFN-α binding molecules provided herein.

For instance, the exemplary anti-IFN-α antibody 19D11 of the presentinvention binds to at least human IFN-α subtypes IFN-α1/13, IFN-α2,IFN-α4, IFN-α5, IFN-α6, IFN-α8, IFN-α10,IFN-α14, IFN-α16 and IFN-α21(see, e.g., FIGS. 13 and 18 to 27) and has neutralization properties asdescribed in the Examples and in FIGS. 5 to 7. Accordingly, in oneembodiment, the antibody or IFN-α binding fragment of the presentinvention in addition to the IFN-α subtypes defined in section (i),supra, binds to at least human IFN-α subtypes IFN-α1/13, IFN-α8, IFN-α16and IFN-α21. Thus, in one preferred embodiment, the anti-IFN-α antibodyor an IFN-α binding fragment, synthetic or biotechnological derivativethereof has the immunological characteristics and/or biologicalproperties of antibody 19D11 as illustrated in the appended Examples andFigures, in particular with respect to its neutralizing activity towardsthe different IFN-α subtypes.

Exemplary anti-IFN-α antibodies 26B9 and 3 1 B4 of the present inventionbind to at least human IFN-α subtypes IFN-α1/13, IFN-α2, IFN-α4, IFN-α5,IFN-α6, IFN-α8, IFN-α10, IFN-α14 and IFN-α21 (see, e.g., FIGS. 13 and 18to 27) and have neutralization properties as described in the Examplesand in FIGS. 5 to 7. Therefore, in one embodiment, the antibody or IFN-αbinding fragment of the present invention in addition to the IFN-αsubtypes defined in section (i), supra, binds to at least human IFN-αsubtypes IFN-α1/13, IFN-α8 and IFN-α21. Thus, in another preferredembodiment, the anti-IFN-α antibody or an IFN-α binding fragment,synthetic or biotechnological derivative thereof has the immunologicalcharacteristics and/or biological properties of antibody 26B9 or 31B4 asillustrated in the appended Examples and Figures, in particular withrespect to its neutralizing activity towards the different IFN-αsubtypes.

Exemplary anti-IFN-α antibody 25C3 of the present invention binds to atleast human IFN-α subtypes IFN-α1/13, IFN-α2, IFN-α4, IFN-α5, IFN-α6,IFN-α8, IFN-α10, IFN-α14, IFN-α16 and IFN-α21 (see, e.g., FIGS. 13 and18 to 19) and has neutralization properties as described in the Examplesand in FIGS. 5 to 7, showing in particular a reduced neutralizationcapability towards IFN-α6, IFN-α8, IFN-α10 and IFN-α16. In oneembodiment thus, the antibody or IFN-α binding fragment of the presentinvention in addition to the IFN-α subtypes defined in section (i),supra, binds to at least human IFN-α subtypes IFN-α1/13, IFN-α8, IFN-α16and IFN-α21 but shows only weak neutralization of IFN-α16. In a furtherembodiment, the antibody or IFN-α binding fragment of the presentinvention shows in addition only weak neutralization of IFN-α6. IFN-α8and IFN-α10. Thus, in another preferred embodiment, the anti-IFN-αantibody or an IFN-a binding fragment, synthetic or biotechnologicalderivative thereof has the immunological characteristics and/orbiological properties of antibody 25C3 as illustrated in the appendedExamples and Figures, in particular with respect to its neutralizingactivity towards the different IFN-α subtypes.

Exemplary anti-IFN-α antibody 5D1 of the present invention binds to atleast human IFN-α subtypes IFN-α2, IFN-α4, IFN-α5, IFN-α6, IFN-α8,IFN-α10, IFN-α14, IFN-α16 and IFN-α21 but not to IFN-α1/13 and hasneutralization properties as described in the Examples and in FIGS. 5 to7. Accordingly, in one embodiment, the antibody or IFN-α bindingfragment of the present invention in addition to the IFN-α subtypesdefined in section (i), supra, binds to at least human IFN-α subtypesIFN-α8, IFN-α16 and IFN-α21 but not to IFN-α1/13. Thus, in anotherpreferred embodiment, the anti-IFN-α antibody or an IFN-α bindingfragment, synthetic or biotechnological derivative thereof has theimmunological characteristics and/or biological properties of antibody5D1 as illustrated in the appended Examples and Figures, in particularwith respect to its neutralizing activity towards the different IFN-αsubtypes.

Exemplary anti-IFN-α antibody 13B11 of the present invention binds to atleast human IFN-α subtypes IFN-α1/13, IFN-α2, IFN-α4, IFN-α5, IFN-α6,IFN-α10. IFN-α14 and IFN-α16 but not to IFN-α8 and has neutralizationproperties as described in the Examples and in FIGS. 5 to 7. Therefore,in one embodiment, the antibody or IFN-α binding fragment of the presentinvention in addition to the IFN-α subtypes defined in section (i),supra, binds to at least human IFN-α subtypes IFN-α1/13 and IFN-α1 6 butnot to IFN-α8. Thus, in a further preferred embodiment, the anti-IFN-αantibody or an IFN-α binding fragment, synthetic or biotechnologicalderivative thereof has the immunological characteristics and/orbiological properties of antibody 13B11 as illustrated in the appendedExamples and Figures, in particular with respect to its neutralizingactivity towards the different IFN-α subtypes.

Exemplary anti-IFN-α antibody 8H1 of the present invention binds to atleast human IFN-α subtypes IFN-α1, IFN-α4, IFN-α5, IFN-α6, IFN-α7,IFN-α10, IFN-α16, IFN-α17 and IFN-α21 while showing weakerneutralization of IFN-α2, IFN-α8 and IFN-α14 and has neutralizationproperties as described in the Examples and in FIG. 7. Therefore, in oneembodiment, the antibody or IFN-α binding fragment of the presentinvention binds in addition to the IFN-α subtypes defined in section(i), supra, to at least human IFN-α subtypes IFN-α1/13, IFN-α17 andIFN-α21. Thus, in a still further preferred embodiment, the anti-IFN-αantibody or an IFN-α binding fragment, synthetic or biotechnologicalderivative thereof has the immunological characteristics and/orbiological properties of antibody 8H1 as illustrated in the appendedExamples and Figures, in particular with respect to its neutralizingactivity towards the different IFN-α subtypes.

Exemplary anti-IFN-α antibody 12H5 of the present invention binds to andneutralizes all human IFN-α subtypes, namely IFN-α1, IFN-α2, IFN-α4,IFN-α5, IFN-α6, IFN-α7, IFN-α8, IFN-α10, IFN-α14, IFN-α16, IFN-α17 andIFN-α21 and has neutralization properties as described in the Examplesand in FIG. 7. Therefore, in one embodiment, the antibody or IFN-abinding fragment of the present invention binds to all human IFN-αsubtypes. Thus, in a still further preferred embodiment, the anti-IFN-αantibody or an IFN-α binding fragment, synthetic or biotechnologicalderivative thereof has the immunological characteristics and/orbiological properties of antibody 12H5 as illustrated in the appendedExamples and Figures, in particular with respect to its neutralizingactivity towards the different IFN-α subtypes.

In one embodiment, the anti-IFN-α antibody and IFN-α binding fragment ofthe present invention recognize and/or neutralize IFN-α21, preferablywith at least substantially the same binding preference/neutralizingactivity as for any other IFN-α recognized and/or neutralized by theantibody or fragment thereof.

Furthermore, after preliminary results obtained in the experimentsperformed within the scope of the present invention already indicatedthat exemplary antibodies provided by the present invention bind inaddition to at least one IFN-α subtype to another type I interferon,i.e. IFN-omega (also indicated as IFN-ω or as IFN-ω herein) asexemplarily shown for antibodies 26B9 and 31B4 in FIG. 18, furtherexperiments confirmed the surprising neutralizing properties of some ofthe subject antibodies towards IFN-ω, i.e. for antibodies 26B9 and 31B4in FIG. 5 and for antibody 26B9 in comparison to antibody 19D11 in FIG.24, for antibody 26B9 in FIGS. 25-26, for antibody 31B4 in FIG. 25, forantibody 8H1 in FIGS. 7 and 20 and for antibody 50E11 in FIG. 22.Accordingly, in one embodiment the present invention also relates toanti-IFN-a antibodies and IFN-α binding fragments thereof which besidesat least one IFN-α subtype bind in addition at least one other type Iinterferon, wherein preferably the other type I interferon is humanIFN-ω (IFN-ω). The corresponding IFN-binding molecules may also bedenoted IFN-α/IFN-ω binding molecule or anti-IFN-α/IFN-ω antibody.Preferably, the anti-IFN-α antibody or IFN-α binding fragment of thepresent invention, in addition to the at least one IFN-a subtype iscapable of neutralizing a biological activity of human IFN-ω (IFN-ω).Such an antibody is of particular value for use in the prevention,treatment or diagnosis of patients having a higher titer of IFN-ω and/orfor patients showing both, an increased titer of an IFN-α subtype and ofIFN-ω. Thus, in a still further preferred embodiment, the anti-IFN-αantibody or an IFN-α binding fragment, synthetic or biotechnologicalderivative thereof has the immunological characteristics and/orbiological properties of antibody 26B9, 31B4, 8H1 or 50E11 asillustrated in the appended Examples and Figures, in particular withrespect to its neutralizing activity towards IFN-ω.

The IFN-α/IFN-ω binding molecules of the present invention areparticularly useful in the prevention, therapy and/or diagnosis ofdiseases associated with an increased expression or level of activity ofIFN-ω in a subject, in particular in case the expression or level ofactivity of IFN-α is increased as well. For example, IFN-ω is found inthe serum of patients with systemic lupus erythematosus (SLE) and renaldisease in addition to elevated levels of IFN-α subtypes; see, e.g., M CDall'Era et al., Ann. Rheum. Dis. 64 (2005), 1692-1697 and Han et al.,Genes and Immunity 4 (2003), 177-186. Accordingly, the IFN-α/IFN-ωbinding molecules of the present invention broadly blocking multipletype I IFNs may be more advantageous as a potential therapeutic agentthan an antibody specific for IFN-α only. In addition, it has beenreported that sera of patients with rheumatoid arthritis besides IFN-have an elevated level of IFN-ω relative to normal controls, but not ofIFN-α; see Lavoie et al., J. Immunol. 186 (2011), 186, meeting abstractio1.37. Thus, the IFN-α/IFN-ω binding molecules of the present inventionmay also prove useful in the treatment of autoimmune disorders which donot or not significantly involve IFN-α but IFN-ω. Hence, in a furtheraspect the present invention relates to IFN-ω binding molecules whichare synthetic or biotechnological derivatives of the human-derivedanti-IFN-α antibody 26B9, 31B4, 8H1 or 50E11 which substantially retaintheir neutralizing activity towards IFN-ω, but lost their bindingspecificity to one or more IFN-α subtypes or the capability of bindingIFN-α altogether. In addition, the present invention relates to anyIFN-ω binding molecule, preferably antibody or antibody fragment whichcompetes with any of the human-derived anti-IFN-α antibodies 26B9, 31B4,8H1 or 50E11 for binding and/or neutralizing IFN-ω in any of the assaysdescribed in the Examples but not necessarily with respect to bindingand/or neutralizing IFN-α.

Furthermore, as shown in FIG. 18, the exemplary anti-IFN-α antibodies ofthe present invention display a much weaker or no affinity towards typeI interferon IFN-ε, IFN-β, towards IFN-γ as a type II interferon and/ortowards IFN-λ (IL-29, IL-28A and IL-28B in FIG. 18) as type IIIinterferons. Accordingly, in one embodiment the present inventionrelates to anti-IFN-a antibodies and IFN-α binding fragments thereofwhich preferably bind type I over type II and/or type III interferons,more preferably anti-IFN-α antibodies and IFN-α binding fragments of thepresent invention do not substantially recognize type II and type IIIinterferons.

The present invention exemplifies IFN-α binding molecules, i.e.antibodies and IFN-α binding fragments thereof, which may be generallycharacterized by comprising in their variable region, i.e. bindingdomain at least one complementarity determining region (CDR) of the VHand/or VL of the variable region comprising the amino acid sequencedepicted in FIG. 1 of (VH) (SEQ ID NOs: 2, 10, 18, 22, 30, 38, 76, 84and 92) and (VL) (SEQ ID NOs: 4, 12, 20, 24, 32, 40, 78, 86 and 94)—seethe exemplary CDR sequences underlined in FIG. 1 and identified inTable 1. However, as discussed in the following, the person skilled inthe art is well aware of the fact that in addition or alternatively CDRsmay be used, which differ in their amino acid sequence from thoseindicated in FIG. 1 by one, two, three or even more amino acids, inparticular in case of CDR2 and CDR3.

In respect of the particular binding preferences and neutralizationabilities towards specific IFN-a subtypes and IFN-ω as shown in theExamples and Figures, the general characterization provided above may besubdivided into the following groups of IFN-α binding molecules, i.e.antibodies and binding fragments thereof of the present invention.

In one embodiment, the antibody or IFN-α binding fragment of the presentinvention is a human-derived antibody and binds to at least one IFN-αsubtype selected from the group consisting of IFN-α1/13, IFN-α2, IFN-α4,IFN-α5, IFN-α6, IFN-α8, IFN-α10, IFN-α14, IFN-α16 and IFN-α21,comprising in its variable region:

-   (a) at least one complementarity determining region (CDR) of the VH    and/or VL variable region amino acid sequences depicted in FIG. 1    (VH) (SEQ ID NO: 18); and FIG. 1 (VL) (SEQ ID NO: 20);-   (b) an amino acid sequence of the VH and/or VL region as depicted in    FIG. 1;-   (c) least one CDR consisting of an amino acid sequence resulted from    a partial alteration of any one of the amino acid sequences of (a);    and/or-   (d) heavy chain and/or light variable region comprising an amino    acid sequence resulted from a partial alteration of the amino acid    sequence of (b).

In addition, or alternatively, the antibody or IFN-α binding fragment ofthe present invention is characterized by displaying the bindingcharacteristics of exemplary antibody 19D11.

As illustrated in Example 5 and shown in FIG. 27A/B, epitopes in IFN-α2specifically recognized by exemplary antibody 19D11 have beenidentified. Accordingly, in one embodiment, the antibody or IFN-αbinding fragment, synthetic or biotechnological derivative of thepresent invention is capable of binding an epitope in IFN-α2 consistingof the amino acid sequence SAAWDETLLDKFYTEL YQ (SEQ ID NO: 99) and/orRITLYLKEKKYSPCAWEV (SEQ ID NO: 100).

In another embodiment, the antibody or IFN-α binding fragment of thepresent invention is a human-derived antibody and binds to at least oneIFN-α subtype selected from the group consisting of IFN-α1/13, IFN-α2,IFN-α4, IFN-α5, IFN-α6, IFN-α8, IFN-α10, IFN-α14 and IFN-α21, comprisingin its variable region: at least one complementarity determining region(CDR) of the VH and/or VL variable region amino acid sequences depictedin FIG. 1 (VH) (SEQ ID NO: 30 or SEQ ID NO:38); and FIG. 1 (VL) (SEQ IDNO: 32 or SEQ ID NO: 40);

-   (a) an amino acid sequence of the VH and/or VL region as depicted in    FIG. 1;-   (b) at least one CDR consisting of an amino acid sequence resulted    from a partial alteration of any one of the amino acid sequences of    (a); and/or-   (c) a heavy chain and/or light variable region comprising an amino    acid sequence resulted from a partial alteration of the amino acid    sequence of (b).

In addition, or alternatively, the antibody or IFN-α binding fragment ofthe present invention is characterized by displaying the bindingcharacteristics of exemplary antibody 26B9 or 31B4. Thus, in thisembodiment the anti-IFN-α antibody or IFN-α binding fragment preferablyalso recognizes and neutralizes IFN-ω; see also supra.

As illustrated in Example 5 and shown in FIG. 27C/D, epitopes in IFN-α2and IFN-ω specifically recognized by exemplary antibody 26B9 have beenidentified. Accordingly, in one embodiment, the antibody or IFN-αbinding fragment, synthetic or biotechnological derivative of thepresent invention is capable of binding an epitope of IFN-α2 consistingof the amino acid sequence YTELYQQLNDLEACVIQG (SEQ ID NO: 101) and/or anepitope of IFN-αW consisting of the amino acid sequenceTG1HQQLQHLETCLLQVV (SEQ ID NO: 102).

In another embodiment, the antibody or IFN-α binding fragment of thepresent invention is a human-derived antibody and binds to at least oneIFN-α subtype selected from the group consisting of subtypes IFN-α1/13,IFN-α2, IFN-α4, IFN-α5, IFN-α6, IFN-α8, IFN-α10, IFN-α14, IFN-α16 andIFN-α21, comprising in its variable region:

-   (a) at least one complementarily determining region (CDR) of the VH    and/or VL variable region amino acid sequences depicted in FIG. 1    (VH) (SEQ ID NO: 22); and FIG. 1 (VL) (SEQ ID NO: 24);an amino acid    sequence of the VH and/or VL region as depicted in FIG. 1;-   (b) at least one CDR consisting of an amino acid sequence resulted    from a partial alteration of any one of the amino acid sequences of    (a); and/or-   (c) a heavy chain and/or light variable region comprising an amino    acid sequence resulted from a partial alteration of the amino acid    sequence of (b).

In addition, or alternatively, the antibody or IFN-α binding fragment ofthe present invention is characterized by displaying the bindingcharacteristics of exemplary antibody 25C3.

In another embodiment, the antibody or IFN-α binding fragment of thepresent invention is a human-derived antibody and binds to at least oneIFN-α subtype selected from the group consisting of subtypes IFN-α2,IFN-α4, IFN-α5, IFN-α6, IFN-α8, IFN-α10, IFN-α14, IFN-α16 and IFN-α21but not to IFN-α1/13, comprising in its variable region:

-   (a) at least one complementarity determining region (CDR) of the VH    and/or VL, variable region amino acid sequences depicted in FIG. 1    (VH) (SEQ ID NO: 2); and FIG. 1 (VL) (SEQ ID NO: 4);-   (b) an amino acid sequence of the Vii and/or VL region as depicted    in FIG. 1;-   (c) least one CDR consisting of an amino acid sequence resulted from    a partial alteration of any one of the amino acid sequences of (a);    and/or-   (d) heavy chain and/or light variable region comprising an amino    acid sequence resulted from a partial alteration of the amino acid    sequence of (b).

In addition, or alternatively, the antibody or IFN-α binding fragment ofthe present invention is characterized by displaying the bindingcharacteristics of exemplary antibody 5D1.

In another embodiment, the antibody or IFN-α binding fragment of thepresent invention is a human-derived antibody and binds to at least oneIFN-α subtype selected from the group consisting of subtypes IFN-α1/13,IFN-α2, IFN-α4, IFN-α5, IFN-α6, IFN-α10, IFN-α14 and IFN-α16, comprisingin its variable region:

-   (a) at least one complementarity determining region (CDR) of the VH    and/or VL, variable region amino acid sequences depicted in FIG. 1    (VH) (SEQ ID NO: 10); and FIG. 1 (VL) (SEQ ID NO: 12);-   (b) an amino acid sequence of the VH and/or VL region as depicted in    FIG. 1;-   (c) least one CDR consisting of an amino acid sequence resulted from    a partial alteration of any one of the amino acid sequences of (a);    and/or-   (d) heavy chain and/or light variable region comprising an amino    acid sequence resulted from a partial alteration of the amino acid    sequence of (b).

In addition, or alternatively, the antibody or IFN-α binding fragment ofthe present invention is characterized by displaying the bindingcharacteristics of exemplary antibody 13B11.

In another embodiment, the antibody or IFN-α binding fragment of thepresent invention is a human-derived antibody and binds to at least oneIFN-α subtype selected from the group consisting of IFN-α1, IFN-α4,IFN-α5, IFN-α6, IFN-α10, IFN-α16, IFN-α17 and IFN-α21, comprising in itsvariable region:

-   (a) at least one complementarity determining region (CDR) of the VH    and/or VL variable region amino acid sequences depicted in FIG. 1    (VH) (SEQ ID NO: 76); and FIG. 1 (VL) (SEQ ID NO: 78);-   (b) an amino acid sequence of the V_(H) and/or V_(L) region as    depicted in FIG. 1;-   (c) at least one CDR consisting of an amino acid sequence resulted    from a partial alteration of any one of the amino acid sequences of    (a); and/or-   (d) a heavy chain and/or light variable region comprising an amino    acid sequence resulted from a partial alteration of the amino acid    sequence of (b).

In addition, or alternatively, the antibody or IFN-α binding fragment ofthe present invention is characterized by displaying the bindingcharacteristics of exemplary antibody 8H1. Thus, in this embodiment theanti-IFN-α antibody or IFN-α binding fragment preferably also recognizesand neutralizes IFN-ω; see also supra.

In another embodiment, the antibody or IFN-α binding fragment of thepresent invention is a human-derived antibody and binds to at least oneIFN-α subtype selected from the group consisting of IFN-α1/13, IFN-α2,IFN-α4, IFN-α5, IFN-α6, IFN-α8, IFN-α10, IFN-α14 and IFN-α21, comprisingin its variable region:

-   (a) at least one complementarity determining region (CDR) of the VH    and/or VL variable region amino acid sequences depicted in FIG. 1    (VH) (SEQ ID NO: 84); and FIG. 1 (VL) (SEQ ID NO: 86);-   (b) an amino acid sequence of the VH and/or VL region as depicted in    FIG. 1;-   (c) at least one CDR consisting of an amino acid sequence resulted    from a partial alteration of any one of the amino acid sequences of    (a); and/or-   (d) heavy chain and/or light variable region comprising an amino    acid sequence resulted from a partial alteration of the amino acid    sequence of (b).

In addition, or alternatively, the antibody or IFN-α binding fragment ofthe present invention is characterized by displaying the bindingcharacteristics of exemplary antibody 12H5.

In another embodiment, the antibody or IFN-α binding fragment of thepresent invention is a human-derived antibody and binds to at least oneIFN-α subtype selected from the group consisting of IFN-α1/13, IFN-α2,IFN-α4, IFN-α5, IFN-α6, IFN-α8, IFN-α10, IFN-α14 and IFN-α21, comprisingin its variable region:

-   (a) at least one complementarity determining region (CDR) of the VH    and/or VL variable region amino acid sequences depicted in FIG. 1    (VH) (SEQ ID NO: 92); and FIG. 1 (VL) (SEQ ID NO: 94);-   (b) an amino acid sequence of the Vi and/or VL region as depicted in    FIG. 1;-   (c) at least one CDR consisting of an amino acid sequence resulted    from a partial alteration of any one of the amino acid sequences of    (a); and/or-   (d) a heavy chain and/or light variable region comprising an amino    acid sequence resulted from a partial alteration of the amino acid    sequence of (b).

In addition, or alternatively, the antibody or IFN-α binding fragment ofthe present invention is characterized by displaying the bindingcharacteristics of exemplary antibody SOE11. Thus, in this embodimentthe anti-IFN-α antibody or IFN-α binding fragment preferably alsorecognizes and neutralizes IFN-ω at least to some extent; see alsosupra.

In summary, in one aspect the present invention relates to human-derivedmonoclonal antibodies and IFN-α binding fragments as well as syntheticand biotechnological derivatives of the subject antibodies exemplifiedherein comprising in their variable regions:

-   (a) at least one complementarity determining region (CDR) of the VH    and/or VL variable region amino acid sequences depicted in    -   (i) FIG. 1 (VH) (SEQ ID NOs: 2, 10, 18, 22, 30, 38, 76, 84 and        92); and    -   (ii) FIG. 1 (VL) (SEQ ID NOs: 4, 12, 20, 24, 32, 40, 78, 86 and        94);-   (b) an amino acid sequence of the VH and/or VL region as depicted in    FIG. 1;-   (c) at least one CDR consisting of an amino acid sequence resulted    from a partial alteration of any one of the amino acid sequences of    (a); and/or-   (d) a heavy chain and/or light variable region comprising an amino    acid sequence resulted from a partial alteration of the amino acid    sequence of (b);

preferably wherein the antibody, IFN-α binding fragment or synthetic andbiotechnological derivative thereof retains at least one of theimmunological characteristics and/or biological activities of any of thesubject antibodies described in the Examples, e.g. neutralizing activitytowards different IFN-α subtypes and/or IFN-ω.

In addition, in one aspect the present invention relates to an IFN-αneutralizing antibody or IFN-α binding fragment thereof which competeswith said antibody or fragment thereof as defined hereinabove forbinding to and/neutralizing of at least one IFN-α subtype and/or IFN-ω.Furthermore, in one embodiment the antibody of the present invention orthe IFN-α binding fragment thereof contains at least CDR3 of the VHand/or VL variable region depicted in

-   -   (i) FIG. 1 (VH) (SEQ ID NOs: 2, 10, 18, 22, 30, 38, 76, 84 and        92); and    -   (ii) FIG. 1 (VL) (SEQ ID NOs: 4, 12, 20, 24, 32, 40, 78, 86 and        94);

or a corresponding CDR3 which differs in its amino acid sequence bysubstitution, deletion and/or addition of 6, 5 or 4, preferably not morethan 3 and most preferably red no more than 2 or 1 amino acids.

In order to provide antibodies particularly suitable for therapeuticapplications, i.e. to avoid immunological responses to the antibodies ofthe present invention as observed for foreign antibodies such as mouseantibodies in humans (RAMA-response) the present invention preferablyrelates to fully human or human-derived antibodies since the exemplaryIFN-α antibodies, which are described in the Examples illustrating thepresent invention, have been derived from a human patient.

In this context, contrary to humanized antibodies and otherwisehuman-like antibodies, see also the discussion infra, the human-derivedantibodies of the present invention are characterized by comprising CDRswhich have been seen by the human body and therefore are substantiallydevoid of the risk of being immunogenic. Therefore, the antibody of thepresent invention may still be denoted human-derived if at least one,preferably two and most preferably all three CDRs of one or both thevariable light and heavy chain of the antibody are derived from thehuman antibodies illustrated herein.

The human-derived antibodies of the present invention may also be called“human auto-antibodies” in order to emphasize that those antibodies wereindeed expressed initially by a human subject and are not in vitroselected constructs generated, for example, by means of humanimmunoglobulin expressing phage libraries or xenogeneic antibodiesgenerated in a transgenic animal expressing part of the humanimmunoglobulin repertoire, which hitherto represented the most commonmethod for trying to provide human-like antibodies. On the other hand,the human-derived antibody of the present invention may be denotedsynthetic, recombinant, and/or biotechnological in order to distinguishit from human serum antibodies per se, which may be purified via proteinA or affinity column.

However, the present invention uses and envisages further studies of theantibodies of the present invention in animal models, e.g., intransgenic mice expressing human IFN-α. To avoid immunogenic effects inthe experimental animals analogous to the RAMA-response in humans, inone aspect, the antibody of the present invention may be a humanized,xenogeneic, or chimeric human-murine antibody, preferably a chimericrodent-human or a rodentized antibody, most preferred a chimericmurine-human or murinized antibody.

As described herein below in more detail, the antibody orantigen-binding fragment thereof of the present invention can be of orderived from any type, class or subclass of an immunoglobulin molecule.However, in a preferred embodiment, the antibody of the presentinvention is provided, which is of the IgG isotype, most preferably ofthe IgG1 subclass.

In order to provide such humanized, chimeric and in particular fullyhuman antibodies, fragments and/or native Fab fragments thereof, theantibody of the present invention preferably further comprises a CHand/or CL constant region comprising an amino acid sequence selectedfrom the CH and CL amino acid sequences set forth in Table 1 (SEQ IDNOs.: 6, 8, 14, 16, 26, 28, 34, 36, 42, 44, 72, 74, 80, 82, 88, 90, 96and 98) or an amino acid sequence with at least 60 identity, preferably70% identity, more preferably 80% identity, still more preferably 90%identity, and particularly preferred at least 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99%, identity to the mentioned reference sequences.

In addition, or alternatively, the framework region of the antibody ofthe present invention comprises an amino acid sequence selected fromFIG. 1 (VH) (SEQ ID NOs: 2, 10, 18, 22, 30, 38, 76, 84 and 92); and FIG.1 (VL) (SEQ ID NOs.: 4, 12, 20, 24, 32, 40, 78, 86 and 94) or an aminoacid sequence with at least 60% identity, preferably 70% identity, morepreferably 80% identity, still more preferably 90% identity, andparticularly preferred at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%or 99%, identity to the mentioned reference sequences.

As mentioned above, the antibodies of the present invention have beenisolated from APECED/APS1 patients. In this context, experimentsdisclosed in applicant's co-pending international applicationWO2013/098419 surprisingly revealed that APECED/APS1 patients display anauto-immunosome, i.e. an autoantibody profile comprising as well a broadspectrum of binding molecules specific for different IFNα subtypes. APS1is a rare autoimmune disease caused by mutations in the AutoimmuneRegulator (AIRE) gene. The AIRE protein governs the expression inmedullary thymic epithelium of many peripheral self-antigens (e.g.,insulin) that are presented by MHC to tolerate developing thymocytes. InAPS1, AIRE mutations cause aberrant negative selection, which enablesautoreactive T cells to escape to the periphery. Accordingly, thepatients show an extremely variable spectrum of clinical features inAPS1, but usually with several autoimmune disorders of endocrinetissues. The defining APS1 triad comprises chronic mucocutaneouscandidiasis, hypoparathyroidism and adrenal failure (Perheentupa,Endocrinol. Metab. Clin. North Am. 31 (2002), 295-320).

Other clinical conditions seen in APECED patients include thyroidautoimmune diseases, diabetes mellitus, gonadal failure, vitiligo,alopecia, chronic hepatitis, chronic gastritis and pernicious anemia anddifferent forms other gastrointestinal symptoms. For further detailsconcerning APECED/APS1 patients and the screening of theirauto-immunosome see the description of international applicationWO2013/098419 and the Examples described therein, in particular theMaterial and Methods section on pages 112-117; Example 1 on pages117-118 and Example 7 on page 128 and the following Tables 1 to 14; andExample 17 on pages 168-171, the disclosure content of which isincorporated herein by reference.

As described in detail above and indicated in Example 1, in onepreferred embodiment the antibody of the present invention is obtainedfrom a sample of a human subject affected with autoimmunepolyendocrinopathy-candidiasis-ectodermal dystrophy (APECED/APS1) orfrom a patient affected with a similar autoimmune disease as describedin international application WO2013/098419 and the Examples therein, inparticular the Materials and Methods section on pages 112-117; Example 1on pages 117-118; in Example 10 on pages 156-161, specifically insection “Patients and controls” on page 156 therein; and Example 17 onpages 168-171, the disclosure content of which is incorporated herein byreference.

In this context it is noted that the subject anti-IFN-α antibodies ofthe present invention have been cloned by a novel and proprietary methodof isolating human antibodies, which is disclosed in applicant'sco-pending international application WO2013/098420, the disclosurecontent of which is incorporated herein by reference.

Briefly, the sample for isolating the antibody of interest comprises orconsists of peripheral blood mononuclear cells (PBMC) and serum for thedetection of possible antibody reactivities. The sample derived from thesubject may either be directly used for, e.g., testing seroreactivityagainst one or more of the desired antigen(s) or may be furtherprocessed, for example enriched for B lymphocytes. In particular, it ispreferred that the sample comprises or is derived from B cells thatproduce the antibody of interest, most preferably memory B-cells. Thememory B cells are cultured under conditions allowing only a definitelife span of the B cells, typically no more than 1 to 2 weeks untilsingling out the cells from B cell cultures which are reactive againstthe desired antigen subsequently followed by RT-PCR of single sortedcells for obtaining the immunoglobulin gene repertoire; see for detaileddescription Examples 1 and 2 on pages 118 to 120 of WO2013/098419 and inparticular Examples 1 to 4 on pages 27 to 31 of WO2013/098420, thedisclosure content of which is incorporated herein by reference.Naturally, the present invention extends to the immortalized human Bmemory lymphocyte and B cell, respectively, that produces the antibodyhaving the distinct and unique characteristics as defined herein aboveand below.

Thus, besides using a selected patient pool, the anti-IFN-α antibodieshave been provided by employing a particular method specificallydeveloped and adapted for isolating human monoclonal antibodies from Bcells of patients with an autoimmune disease such as APECED/APS1patients.

In one embodiment, the antibody or IFN-α binding molecule of the presentinvention comprises an amino acid sequence of the VH and/or VL region asdepicted in FIG. 1 or as encoded by the corresponding nucleic acids asindicated in Table 1. In addition, in another embodiment the presentinvention relates to an anti-IFN-α antibody or IFN-α binding molecule,which competes with an antibody of the present invention as definedhereinabove for specific binding to at least one human IFN-α subtypeand/or human IFN-ω.

In particular, anti-IFN-α antibodies are provided which demonstrate theimmunological binding characteristics and/or biological properties asoutlined for the antibodies illustrated in the Examples and in theFigures. Where present, the term “immunological bindingcharacteristics,” or other binding characteristics of an antibody withan antigen, in all of its grammatical forms, refers to the specificity,affinity, cross-reactivity, and other binding characteristics of anantibody.

As demonstrated in the Examples, the antibodies of the present inventionare particularly characterized by their high neutralizing activitytowards the majority of the IFN-α subtypes as well as in case of the26B9, 31B4 and 8H1 antibody against IFN-ω in the sub-nanomolar range.Preferably, the human-derived monoclonal antibodies and IFN-α bindingfragments as well as synthetic and biotechnological derivatives thereofdisplay the IC50 values as determined in an ISRE (Interferon stimulatedresponse element) reporter gene assay of the subject antibodiesexemplified herein for at least one, two, three, four, five, six, seven,eight, nine, ten, eleven, twelve or almost all IFN-α subtypes andoptionally for IFN-ω or their IC50 values differ no more than 50%,preferably less than 40%, more preferably less than 30%, still morepreferably less than 20% and particularly preferred less than 10% fromthe IC50 values determined for the subject antibodies illustrated in theExamples and Figures for any of the IFN-α subtypes and optionally forIFN-ω. In a preferred embodiment, the antibody or like IFN-bindingmolecule of the present invention has an IC 50 value of :S 10 ng for atleast 5, preferably 6, more preferably 7, still more preferably 8 oradvantageously 9 or 10 human IFN-α subtypes and/or human IFN-ω.

In a further embodiment, the antibody of the present invention is anantibody fragment. For example, the antibody or antibody fragment of thepresent invention may be elected from the group consisting of a singlechain Fv fragment (scFv), an F(ab′) fragment, an F(ab) fragment, anF(ab′)2 fragment and a single domain antibody fragment (sdAB).

A further advantage of the antibodies of the present invention is thatdue to the fact that the humoral immune response has been elicitedagainst the native antigen in its physiologic and cellular environment,typically autoantibodies are produced and can be isolated whichrecognize a conformational epitope of the antigen due to itspresentation in context for example with other cellular components,presentation on a cell surface membrane and/or binding to a receptor. Incontrast, conventional methods of generating monoclonal antibodies suchas mouse monoclonals, humanized versions thereof or antibodies obtainedfrom phage display typically employ an antigenic fragment of the targetprotein for immunizing an non-human mammal and detection, respectively,upon which usually antibodies are obtained which recognize linearepitopes or conformational epitopes limited to a two-dimensionalstructure of the immunogen rather than the presence of the nativeprotein in its physiological and cellular context. Accordingly, it isprudent to expect that the autoantibodies of the present invention areunique in terms of their epitope specificity. Therefore, the presentinvention also relates to antibodies and like-binding molecules whichdisplay substantially the same binding specificity as the autoantibodiesisolated in accordance with the method of the present invention. Suchantibodies can be easily tested by for example competitive ELISA or moreappropriately in a cell based neutralization assay using an autoantibodyand a monoclonal derivative, respectively, thereof of the presentinvention as a reference antibody and the immunological tests describedin the Examples or otherwise known to the person skilled in the art.

As further illustrated in the Examples and in the Figures, e.g., in FIG.19, the antibodies of the present invention are isolated preferably fromhuman donors and thus bind human IFN-α subtypes. Therefore, in oneembodiment the anti-IFN-α antibody and IFN-α binding fragment of thepresent invention recognize only the human antigen or at leastpreferentially over the corresponding antigen from other species such asmice. In another embodiment the anti-IFN-α antibody and IFN-α bindingfragment of the present invention bind at least one IFN-α subtypes ofother species, preferably wherein the other species is mice. Bindingcharacteristics such as specificity and affinity of the antibodies ofthe present invention have been tested in several experimental assays asdescribed and shown herein, e.g., in Examples 2, 5 and 6 and in FIGS.2-3 and 8-13, and 18 to 27.

As has been further demonstrated for the antibodies of the presentinvention, they are capable of neutralizing the biological activity oftheir target protein; see, e.g., the results of the STAT1phosphorylation assay and the interferon specific response elementreporter-gene assay described in Example 3 and FIGS. 5 to 7. In thiscontext, the term “neutralizing” means that the antibody of the presentinvention is capable of intervening with the biological activity of itstarget protein in a biochemical or cell-based assay as can be evaluatedby performing the respective assay in the presence of the subjectantibody of the present invention, wherein the biological activity ofthe target protein is reduced concomitantly with increasing level of theantibody of the present invention subjected to the assay compared to thebiological activity of the protein without the presence of the antibodyof the present invention and in the presence of a compound for example acontrol antibody which is known to leave the biological activity of thetarget protein unaffected in kind. Such biochemical and in vitro basedassay can also be performed using a reference antibody known to becapable of neutralizing the biological activity of the target proteinsuch as has been shown for the anti-IFN-α antibodies of the presentinvention and subjecting the candidate antibody to the test sample,wherein either an additive neutralizing effect may be observed resultingfrom the combined activity of the reference and candidate antibody or acompetition of the candidate antibody and reference antibody is observedwhich may be determined by labelling either antibody. Thus, in apreferred embodiment of the present invention, the antibody obtained bythe method of the present invention is capable of neutralizing thebiological activity of its antigen, e.g., at least one human IFN-αsubtype.

The antibodies or antigen-binding fragments, e.g., peptides,polypeptides or fusion proteins of the present invention may beprovided, as indicated above, by expression in a host cell or in an invitro cell-free translation system, for example. To express the peptide,polypeptide or fusion protein in a host cell, the nucleic acid moleculeencoding said peptide, polypeptide or fusion protein may be insertedinto appropriate expression vector, i.e. a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence. Methods which are well known to those skilled in theart may be used to construct expression vectors containing sequencesencoding a polypeptide of interest and appropriate transcriptional andtranslational control elements. These methods include in vitrorecombinant DNA techniques, synthetic techniques, and in vivo geneticrecombination. Such techniques are described in Sambrook et al.,Molecular Cloning, A Laboratory Manual (1989), and Ausubel et al.,Current Protocols in Molecular Biology (1989); see also the sections“Polynucleotides” and “Expressions” further below and literature citedin the Examples section for further details in this respect.

A suitable host cell for expression of the product may be anyprokaryotic or eukaryotic cell; e.g., bacterial cells such as E. coli orB. subtilis, insect cells (baculovirus), yeast cells, plant cell or ananimal cell. For efficient processing, however, mammalian cells arepreferred. Typical mammalian cell lines useful for this purpose includeCHO cells, HEK 293 cells, COS cells and NSO cells.

The isolated antibodies of the present invention may of course not beapplied as such to a patient, but usually have to be pharmaceuticallyformulated to ensure, e.g., their stability, acceptability andbioavailability in the patient. Therefore, in one embodiment, the methodof the present invention is provided, further comprising the step ofadmixing the isolated monoclonal antibody or a fragment thereof with apharmaceutically acceptable carrier. Pharmaceutically acceptablecarriers will be described in detail further below.

As a measure to obtain a stable and permanent source of bindingmolecules of the present invention, in particular for pharmaceutical useheterologous genes encoding these binding molecules may be isolated bydirect cloning, PCR amplification, or artificial synthesis andintroduced and expressed in suitable host cells or organisms. Therefore,it is also an object of the present invention to provide a method forpreparing a recombinant cell useful for the production of a recombinanthuman anti-IFN-α antibody or IFN-α binding fragment thereof, comprisingthe steps of:

-   (a) preparing a B cell by a method as described above;-   (b) sequencing a nucleic acid and/or obtaining from the B cell a    nucleic acid that encodes;    -   (i) at least one of the CH and CL amino acid sequences set forth        in Table 1 (SEQ ID NOs.: 6, 8, 14, 16, 26, 28, 34, 36, 42, 44,        72, 74, 80, 82, 88, 92, 96 and 98) or an amino acid sequence        with at least 60% identity;    -   (ii) at least one complementarity determining region (CDR) of        the VH and/or VL variable region amino acid sequences depicted        in        -   FIG. 1 (VH) (SEQ ID NOs: 2, 10, 18, 22, 30, 38, 76, 84 and            92); and        -   FIG. 1 (VL) (SEQ ID NOs: 4, 12, 20, 24, 32, 40, 78, 86 and            94);    -   (iii) an amino acid sequence of the VH and/or VL region as        depicted in FIG. 1;    -   (iv) at least one CDR consisting of an amino acid sequence        resulted from a partial alteration of any one of the amino acid        sequences of (a);    -   (v) a heavy chain and/or light variable region comprising an        amino acid sequence resulted from a partial alteration of the        amino acid sequence of (ii); and/or-   (c) inserting the nucleic acid into an expression host in order to    permit expression of the antibody of interest in that host.

Host cells as described herein may be used as well in the precedingmethod and as described in detail in the “Host” section of thisspecification. In this respect, in one embodiment the above method isprovided, where the expression host is a yeast cell, a plant cell or ananimal cell.

Furthermore, in one embodiment a method is provided for preparing apharmaceutical composition for use in the treatment of a disorderassociated with the expression and activity of IFN-α and/or IFN-ω, themethod comprising:

-   (a) culturing host cells as defined above;-   (b) purifying the antibody, IFN-α binding fragment thereof,    biotechnological derivative or immunoglobulin chain(s) thereof of    the present invention from the culture to pharmaceutical grade; and-   (c) admixing the antibody, IFN-α binding fragment thereof or    biotechnological derivative thereof of the present invention with a    pharmaceutically acceptable carrier.

In respect of the above described methods for production of therespective antibody of interest, in one embodiment the present inventionprovides a method, wherein the nucleic acid is manipulated between abovesteps (b) and (c) to introduce restriction sites, to change codon usage,and/or to add or optimize transcription and/or translation regulatorysequences. As demonstrated in appended Examples 2 and 3 and summarizedin Table 4, binding molecules, i.e. antibodies have been identified andcloned, which display a particularly high apparent binding affinity(EC50/ED50) and/or a particularly high in vitro neutralizing activitywith low inhibitory concentrations (IC50) for at least one human IFN-αsubtype. In this respect, in one embodiment, the anti-IFN-α antibody andIFN-α binding fragment of the present invention thereof have a highaffinity for its respective target molecule, e.g., human IFN-α subtypesas defined hereinabove, showing an EC50 at concentrations below 100ng/ml, preferably below 20 ng/ml and more preferably below 10 ng/ml.Alternatively or in addition, in one embodiment the anti-IFN-α antibodyand IFN-α binding fragment thereof have high neutralizing ability for atleast one human IFN-α subtype, showing IC50 at concentrations below500,400,300 or 100 ng/ml, preferably below 20 ng/ml, more preferablybelow 10 ng/ml and most preferred below 5 ng/ml. For more details inrespect of the binding affinity of the antibodies of the presentinvention see, e.g., section “Binding characteristics” further below.

The present invention also relates to polynucleotides encoding at leasta variable region of an immunoglobulin chain of the antibody orantigen-binding fragment of the invention. Preferably, said variableregion comprises at least one complementarity determining region (CDR)of the VH and/or V_(L) of the variable region as set forth in FIG. 1.

In case of a derived sequence, said sequence shows at least 60%identity, more preferably (in the following order) at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, and most preferably95%, at least 96-99%, or even 100% identity to a sequence of the groupconsisting of those sequences referred to above and identified in theSequence Listing. The percent identity between two sequences is afunction of the number of identical positions shared by the sequences,taking into account the number of gaps, and the length of each gap,which need to be introduced for optimal alignment of the two sequences.The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm, which is well known to those skilled in the art. Theidentities referred to herein are to be detetinined by using the BLASTprograms as further referred to herein infra.

As mentioned above, in a preferred embodiment, the present inventionrelates to substantially fully human antibodies, preferably IgGincluding at least the constant heavy chain I (CHI) and thecorresponding light chain of the constant region, i.e. y-I, y-2, y-3 ory-4 in combination with lambda or kappa. In a particularly preferredembodiment, the nucleotide and amino acid sequences of those constantregions isolated for the subject antibodies illustrated in the Examplesare used as depicted in Table 1 below and in SEQ ID NOs: 5, 7, 13, 15,25, 27, 33, 35, 41, 43, 71, 73, 79, 81, 87, 89, 95 and 97 in respect ofthe nucleotide sequences and/or SEQ ID NOs: 6, 8, 14, 16, 26, 28, 34,36, 42, 44, 72, 74, 80, 82, 88, 90, 96 and 98 in respect of the aminoacid sequences or amino acid sequences with at least 60% identity tothese referenced before.

In accordance with the above, the present invention also relates to apolynucleotide, in particular recombinant polynucleotide encoding atleast the variable region of one immunoglobulin chain of the anti-IFN-αantibody or IFN-α binding fragment of the present invention. Typically,said variable region encoded by the polynucleotide comprises at leastone complementarity determining region (CDR) of the VH and/or VL of thevariable region of the said antibody. Variable and constant regions ofantibodies are described in more detail in the section “IgG structure”below. In a preferred embodiment of the present invention, thepolynucleotide comprises, consists essentially of, or consists of anucleic acid having a polynucleotide sequence encoding the VH or VLregion of an antibody of the present invention as depicted in Table 1below. In this respect, the person skilled in the art will readilyappreciate that the polynucleotides encoding at least the variabledomain of the light and/or heavy chain may encode the variable domain ofeither immunoglobulin chains or only one of them. In a preferredembodiment, the polynucleotide encodes the anti-IFN-α antibody or IFN-αbinding fragment as defined hereinabove.

TABLE 1 Nucleotide sequences of the variable and constant regions(VH, VL, CH, CL) regions of IgGL, kappa, IFN-α specific5Dl, 13B11, 19Dll, 25C3, 26B9, 31B4, 8Hl, 12H5 and 50Ellantibodies of the present invention. Underlined, bold nucleotidesor amino acids indicate the CDR coding regions in the variablechain sequence. Underlined, italic nucleotides or amino acidsindicate sequences which have not been sequenced butobtained from database. In the constant chains, such regionsare aligned with and tuned in accordance with the pertinenthuman germ line variable region sequences in the database;sec, e.g., Vbase (http://vbase.mrc-cpe.cam.ac.uk) hosted bythe MRC Centre for Protein Engineering (Cambridge, UK).Nucleotide and amino acid sequences of variable heavy (VH) and variable lightAntibody (VL), constant heavy (CH) and constant light (CL) chains.5D1-V_(H)gaagtgcaactggtgcaggccggcgcagaggtgaaagcgcccggggagtctctgaggatctcctgtaaggtgtctggatacacctttaca agttattggatcagttgggtgcgccagattcccgggaaaggcctggagtggatggtg aaaattgatcctagagactcttataccatctacaacccgtccttccaaggccacgtctccatctcagttgacaagtccatcaccactgtctacctgcagtggagcagcctgcaggcctcggacaccgccatttattattgtgtgagacattatcttacacagtcat tggtggactactttgaccactggggccagggaacgctggtcgccgtctcctct SEQ ID NO: 1 5D1-V_(H)EVQLVQAGAEVKAPGESLRISCKVSGYTFT SYWIS WVRQIPGKGLEWMV KIDPRDSYTIYNPSFQGHVSISVDKSITTVYLQWSSLQASDTAIYYCVR HYLTQSLVDYFDH WGOGTLVAVSS SEQ ID NO: 25D1-V_(L)gacattcagatgacccagtctccatcctccctgtctgcatctgtgggagacagtgtcaccatcacttgccgggcaagtc kappa-type agagcgtatccaactacttccattggtatcgacagaagcccgggaaagcccctgaactcctgatctat tctgcatcc aatttgcaaactggggtcccatcaagattcactggcagtgggtctgggacagaatgcactctcaccatcaccagtctgcagcctgatgatttcgcaacttactactgt caacagactcacggttacccgttcacttttggccaggggaccaagctgg acgtcaga SEQ ID NO: 3 5D1-V_(L)DIQMTQSPSSLSASVGDSVTITC RASQSVSNYFH WYRQKPGKAPELLIY SA kappa-type SNLQTGVPSRFTGSGSGTECTLTITSLQPDDFATYYC QQTHGYPFT FGQG TKLDVR SEQ ID NO: 45D1-C_(H)gcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga SEQ ID NO: 5 5D1-C_(H)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK SEQ ID NO: 6 5D1-C_(L)cgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgckappa-typectgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctacgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttc aacaggggagagtgttag  SEQ ID NO: 75D1-C_(L) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG kappa-typeNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTK SFNRGEC SEQ ID NO: 8 13B11-V_(H)Gacgtacagctgttgcagtctgggggaggcttgatacagccgggggggtccctgagactctcctgtgcagcctctggctttacttttaag gactatgccatgagttgggtccgccaggctccagggaagggcctggagtgggtctca gtaataagtcgtagtggtaatattgtagactatgtcgactccgtgaagggccggttcaccgtctccagagacaattccaacaacacactcatctgcaaatggacggcctgagagccgacgacacggccatttattactgtgcgaaacccaaggatatgat tgtcgtggtccctgcgggctttgactcctggggccagggaacccttgtctccgtctcctca SEQ ID NO: 9 13B11-V_(H)DVQLLQSGGGLIQPGGSLRLSCAASGFTFK DYAMS WVRQAPGKGLEWVS VISRSGNIVDYVDSVKGRFTVSRDNSNNTLFLQMDGLRADDTAIYYCAK PKDMIVVVPAGFDSWGQGTLVSVSS SEQ ID NO: 10 13B11-V_(L)gacatccagatgacccagtttccatccaccctgtctgcatctgttggagacagcgtcaccatcacttgccgggccagt kappa-type cagagcattagtgcctggttggcctggtatcagcagaaaccagggaaagcccctaaactcctgatctat aaggggt ctagattagaaaacggggtcccatcgaggttcagcggcagtggatctgggacagaattcactctcaccatcggcagcctgcagcctgatgattttgcaacttattactgc caacaatataagacttggacgttcggccaagggaccaaggtgga aatcaaa SEQ ID NO: 11 13B11-V_(L)DIQMTQFPSTLSASVGDSVTITC RASQSISAWLA WYQQKPGKAPKLLIY kappa-type KGSRLENGVPSRFSGSGSGTEFTLTIGSLQPDDFATYYC QQYKTWT FGQ GTKVEIK SEQ ID NO: 1213B11-C_(H)gcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga SEQ ID NO: 13 13B11-C_(H)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 14 13B11-C_(L)cgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgckappa-typectgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag SEQ ID NO: 15 13B11-C_(L)RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG kappa-typeNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC SEQ ID NO: 1619D11-V_(H) gaggtgcagctgttggagtctggggctgaggtgaagaggcctgggtcgtcggtgagggtctcctgcagggcttctggagacaccttcagc agttaccctatcagttgggtgcgacaggcccctggacaaggccttgagtggatggga aggatcctccctgcccttggtgtcacaaactacgctcagaacttccggggcagaatcacgattaccgcggacaagtcgcccctcacagcctacttggaactgagtagcctcagatttgaggacacggccgtgtattactgtgcgagtcccagtgcgg acataattccttcgattttggggacgaccctctttgccttc tggggccaggggaagcctggtcaccgtctcctca SEQ ID NO: 17 19D11-V_(H) EVQLLESGAEVKRPGSSVRVSCRASGDTFS SYPIS WVRQAPGQGLEWMG RILPALGVTNYAQNFRGRITITADKSPLTAYLELSSLRFEDTAVYYCAS PSADIIPSILGTTLFAF WGQGS LVTVSS SEQ ID NO: 18 19D11-V_(L) gaaattgtgttgacgcagtctccaggcaccctgtctctgtctccgggggaaggggccaccctctcctgc agggccag kappa-typetcagaatgttagcagacactacttaacctggtaccagcagaaacctggccagtctccccggctcctcatctat ggtg gctccagcagggccactggcgtcccagacaggttcagtggcggtgggtctgggacagacttcactctcaccatcagcaggctggagcctgaagactttgcgtgtttactgc cagagctatcatagcccacctcctgtgtacactttcggccag gg gaccaaggtggagatcaaa  SEQ ID NO: 19 19D11-V_(L) EIVLTQSPGTLSLSPGEGATLSC RASQNVSRHYLT WYQQKPGQSPRLLI kappa-type Y GGSSRATGVPDRFSGGGSGTDFTLTISRLEPEDFAVFYC QSYHSPPPV YT FGQ GTKVEIK  SEQ ID NO: 2019D11-C_(H) gcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaanancctctccctgtccccgggtaaatgaSEQ ID NO: 71 19D11-C_(H) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQXXLSLSPGK SEQ ID NO: 72 19D11-C_(L)cgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcckappa-typetgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag SEQ ID NO: 73 19D11-C_(L)RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGN kappa-typeSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC SEQ ID NO: 7425C3-V_(H)gagatgcagctgatggagtctgggggaggtttggtacaaccgggggggtccctgagactctcctgtgtagcctctggtttcacctttaaa agttttgcgatgagttgggtccgccaggctccagggaaggggctggagtgggtcgct agtgtcggctctcagggtggcagcaaatactatgcaccctccgtgaagggccggttctccatctccagagacaattccaacaacactctctatgtgcaaatgaacagcctgggagtcgaggacacggccttttattattgtgttaaagagaccgat gcagtggcgacgatggacgctcttgacatgtggggccaagggaccctggtcatcgtctctacc SEQ ID NO: 21 25C3-V_(H)EMQLMESGGGLNQPGGSLRLSCVASGFTFK SFAMS WVRQAPGKGLEWVA SVGSQGGSKYYAPSVKGRFSISRDNSNNTLYVQMNSLGVEDTAFYYCVK ETDAVATMDALDM WGQGTLVIVST SEQ ID NO: 2225C3-V_(L)gacatccgggtgacccagtctccatcctccctgtctgcatctgtcggagacagggtctccatctcttgccagacaagt kappa-type cagagtgttaacatatatctaaattggtatcaacagagaccagggaaaggccctcagctcctgatctct gctgcttc cactttgcagagtggggtcccatcaaggttcagtggcagtggatctgggacagacttcatcctcaccatcatcagtctacaacctgaagattctgcatcctactactgt caacagggttacattaccccgtacacttttggccaggggaccaaggtg gagatcaaa SEQ ID NO: 23 25C3-V_(L)DIRVTQSPSSLSASVGDRVSISC QTSQSVNIYLN WYQQRPGKGPQLLIS AAS kappa-type TLQSGVPSRFSGSGSGTDFILTIISLQPEDSASYYC QQGYITPYT FGQGTKV EIK SEQ ID NO: 2425C3-C_(H)gcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtccccgggtaaatgaSEQ ID NO: 25 25C3-C_(H)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 26 25C3-C_(L)cgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcckappa-typetgctgaataacctctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaac aggggagagtgttag SEQ ID NO: 27 25C3-C_(L)RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNLYPREAKVQWKVDNALQSG kappa-typeNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKS FNRGEC SEQ ID NO: 28 26B9-V_(H)cagatactactgcaggagtcgggcccaggactggtgaagcccacggagaccctgtccctcacctgtagtgtctctggtgactccatcagt gatagtagtcactactgggcctggattcgccagcccccagggaagggaccagagtggattggc agtgtctattttagttcgatgacccactacaacccgtccctcaaaagtcgcgtcagcatctccgttgacaagcccaagaaccagttctccttaaaagtgacctctgtgactgtcgccgacacggccacatattactgtgcgagacaagcccttgcccga gtcggagccatgaattggttcgacccctggggccagggatctctggtcacagtctcctca SEQ ID NO: 29 26B9-V_(H)QILLQESGPGLVKPTETLSLTCSVSGDSIS DSSHYWA WIRQPPGKGPEWIG SVYFSSMTHYNPSLKSRVSISVDKPKNQFSLKVTSVTVADTATYYCAR QAL ARVGAMNWFDPWGQGSLVTVSS SEQ ID NO: 30 26B9-V_(L)gacatcataatgacccagtctccagactccctgcctgtgtctctgggcgagggggtcaccatcaactgcaagtccagc kappa-type cagagcgtcttttcacctccagtaataagagttgtttagcttggtatcagcagaagccaggaaagtctcccaaattgct catttac tgggcatcaacccgccaatccggggtccctgaccgattcagaggcagcgggtctgggacagatttctctctcaccatcaccagtctgcaggctgaagatgtggctgtttatttctgtcagcagtgtcagacatcccctcccact ttcggcggagggaccaggttggagatcaaa SEQ ID NO: 31 26B9-V_(L) DIIMTQSPDSLPVSLGEGVTINCKSSQSVFFTSSNKSCLA WYQQKPGKSPKL kappa-type LIY WASTRQSGVPDRFRGSGSGTDFSLTITSLQAEDVAVYFC QQCQTSPPT FGGGTRLEIK SEQ ID NO: 3226B9-C_(H)gcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtccccgggtaaatgaSEQ ID NO: 33 26B9-C_(H)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 34 26B9-C_(L)cgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcckappa-typetgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag SEQ 1D NO: 35 26B9-C_(L)RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG kappa-typeNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGISSPVTKS FNRGEC SEQ ID NO: 3631B4-V_(H)cagatacagctcaggagtcgggcccaggactggtgaggcccacggagaccctgtccctcacttgtagtgtctctggtgactccatcagt cagagtagtcattactgggcctggattcgccagcccccagggaagggaccagaatggattggcagtgtctattttagctcgatgacccactacaacccgtccctcacaagtcgcgtcagcatctccattgacaaggccatgaataagttctccttaaaagtgacctctgtgactgtcgccgacacggccacatattactgtgcgagacaggccct tgcccgagtcggagccatgaattggttcgacccctggggccagggatctctggtcacagtctcctca SEQ ID NO: 37 31B4-V_(H)QIQLQESGPGLVRPTETLSLTCSVSGDSIS QSSHYWA WIRQPPGKGPEWIG SVYFSSMTHYNPSLTSRVSISIDKAMNKFSLKVTSVTVADTATYYCAR QAL ARVGAMNWFDPWGQGSLVTVSS SEQ ID NO: 38 31B4-V_(L)gacatcataatgacccagtctccagagtccctgcctgtgtctctgggcgagggggtcaccatcaactgcaagtccagc kappa-type cagagcgtctttttcacctccagtaataggagttgtttagcttggtatcagcagaagccaggacagtctcccaaattgct catttac tgggcatcaacccgccaatccggggtccctgaccgattcacaggcagcgggtctgggacagatttctctctcaccatcgccggtctgcaggttgaagatgtggctgtttatttctgtcagcagtgtcacgcatcccctcccact ttcggcggcgggaccaggttggagctcaga SEQ ID NO: 39 31B4-V_(L) DIIMTQSPESLPVSLGEGVTINCKSSQSVFFTSSNRSCLA WYQQKPGQSPKL kappa-type LIY WASTRQSGVPDRFTGSGSGTDFSLTIAGLQVEDVAVYFC QQCHASPPT FGGGTRLELR SEQ ID NO: 4031B4-C_(H)gcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtccccgggtaaatgaSEQ ID NO: 41 31B4-C_(H)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 42 31B4-C_(L)cgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcckappa-typetgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaaca ggggagagtgttag SEQ ID NO: 43 31B4-C_(L)RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN kappa-typeSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFN RGEC SEQ ID NO: 44 8H1 V_(H)caggtgcagctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttctggacagaccttcacc agtgatgatatcaac tgggtgcgacaggcccctggacagggctagagtggatgggatggagg aaccctaacactcaggacacgggctatgcacagaagttccacggcagactcaccttgaccagcaacagttccataagtacatcctatctggagttgagcggcctgagatctgaggacacggccgtgtattactgtgcgagagcggggacttcgacct tgaccggccactacttcgctttgggggtctggggccaggggaccacggtcatcgtctcctca SEQ ID NO: 75 8H1 V_(H)QVQLVQSGAEVKKPGASVKVSCKASGQTFT SDDIN WVRQAPGQGIEWMG WRNPNTQDTGVAQKFHGRLTLTSNSSISTSYLELSGLRSEDTAVYYCAR AGTSTLTGHYFALGVWGQGTTVIVSS SEQ ID NO: 76 8H1 V_(L)gacatccagctgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgtcaggcgactca kappa-type ggatattagcaaatatttaaattggtatcagcagaaaccagggaaagtccctaaactcctgatctac gaaacatccaa tttggaagtaggggtcccatcaaggttcagtggaagtgggtctgggacacattttactctcaccatcagcagcctgcaggctgaagattttgcaacatattactgt caacagtatgagaatttcccgttcactttcggcggagggaccaaggtggagatc aaa SEQ ID NO: 77 8H1 V_(L)DIQLTQSPSSLSASVGDRVTITC QATQDISKYLN WYQQKPGKVPKLLIY ETS kappa-type NLEVGVPSRFSGSGSGTHFTLTISSLQAEPFATYYC QQYENFPFT FGGGTKV EIK SEQ ID NO: 788H1 C_(H)gcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagagagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtccccgggtaaaSEQ ID NO: 79 8Hl C_(H)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 80 8Hl C_(L)cgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctkappa-typegctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag SEQ ID NO: 81 8Hl C_(L)RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN kappa-typeSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC SEQ ID NO: 8212H5 V_(H)caagtgcaactgatacagtctgggcctgaggtgaagaggcctggggcctcagtgaaggtctcctgcaaggcgtctgaaaacaccttcgac actcattatattaat tgggtgcgacaggcccctggacaagggcttacttggctgggatggctgaac cctaccactggtaaaacaggctttccacaaaagtttaagggcagagtcattctgaccagcgacacctccctaaatactgcctatatggaagtgagccgcctgacatctgaggacacggccgtttatttctgtgccagagttttgaagttgtctgatgagt acaactatggtttcgacgtctggggccaagggaccacggtcatcgtctcctca SEQ ID NO: 83 12H5 V_(H)QVQLIQSGPEVKRPGASVKVSCKASENTFD THYIN WVRQAPGQGLTWLG WLNPTTGKTGFPQKFKGRVILTSDTSLNTAYMEVSRLTSEDTAVYFCAR VLKLSDEYNYGFDVWGQGTTVIVSS SEQ ID NO: 84 12H5 V_(L)gacatccaggtgacccagtctccatcctccctgtctgcatctattggggacagagtcaccatcacgtgccgggcaagtc kappa-type agaacattctcacctttataaattggtatcagcacaaaccagggaaagcccctaaactcctgatctat gctgcatccgtt ttacaaaatgaagtcccatcaaggttcagtggcagtggatctgggacagatttcactctcaccatcaccagtctgcaacctgacgattttggaacttactactgt cagcagacttaccttacccctcaatgcagttttggccaggggaccaaggtggagat caaa SEQ ID NO: 85 12H5 V_(L)DIQVTQSPSSLSASIGDRVTITC RASQNILTFIN WYQHKPGKAPKLLIY AA kappa-type SVLQNEVPSRFSGSGSGTDFTLTITSLQPDDFGTYYC QQTYLTPQCS FGQG TKVEIK SEQ ID NO: 8612H5 C_(H)gcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgaSEQ ID NO: 87 12H5 C_(H)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 88 12H5 C_(L)cgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctkappa-typegctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag SEQ ID NO: 89 12H5 C_(L)RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN kappa-typeSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC SEQ ID NO: 9050E11 V_(H)caggtgcagctggtgcagtctggggcagagatgaagaagcctgggtcctcggtgaaggtctcctgcaaggattttggaggcaccttcagc gtctatggtgtcaac tgggtgcgacaggcccctggacaagggcttgagtggatgggggggctcatc cctgtcattgggccagctaactacgcacagaagttccagggcagaatcaccattactgcggacgaatccacgagcacagcctatatggagttgagcagcctgagatttgacgacacggccatttattattgtgtgagagacgacaacgaatat tgg ggccagggaaccctggtcaccgtctcctcg SEQ ID NO: 9150E11 V_(H) QVQLVQSGAEMKKPGSSVKVSCKDFGGTFS VYGVN WVRQAPGQGLEWM GGLIPVIGPANYAQKFQG RITITADESTSTAYMELSSLRFDDTAIYYC VR DDNEYWGQGTLVTVSS SEQ ID NO: 92 50E11 V_(L)gaaatggtgctgacacagtctccagccaccctgtctttgtctccaggagaaagagccaccctctcctgtagggccagtc kappa-type agactgttagcaccttcttagcctggtaccaacagaaacctggccaggttcccaggctcctcgtctac gatatctcctcc agggccaatggcactccagccaggttcagtggcggtgggtctgggacagacttcactctcaccatcagcagcctagaacttgaagattttgcggtttattactgt cagtggcgtagcaactggcctccctcgctcactttcggcggagggaccagggtg gagatcaaa SEQ ID NO: 93 50E11 V_(L)EMVLTQSPATLSLSPGERATLSC RASQTVSTFLA WYQQKPGQVPRLLVY DI kappa-type SSRANGTPARFSGGGSGTDFTLTISSLELEDFAVYYC QWRSNWPPSLT FGG GTRVEIK SEQ ID NO: 9450E11 C_(H)gcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgagggtctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgaSEQ ID NO: 95 50E11 C_(H)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEGLHNHYTQKSLSLSPGK SEQ ID NO: 96 50E11 C_(L)cgaactgtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctkappa-typegctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag SEQ ID NO: 97 50E11 C_(L)RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN kappa-typeSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC SEQ ID NO: 98

The person skilled in the art will readily appreciate that the variabledomain of the antibody having the above-described variable domain can beused for the construction of other polypeptides or antibodies of desiredspecificity and biological function. Thus, the present invention alsoencompasses polypeptides and antibodies comprising at least one CDR ofthe above-described variable domain and which advantageously havesubstantially the same or similar binding properties as the antibodydescribed in the appended examples. The person skilled in the art willreadily appreciate that using the variable domains or CDRs describedherein antibodies can be constructed according to methods known in theart, e.g., as described in European patent applications EP 0 451 216 A1and EP 0 549 581 A1. Furthermore, the person skilled in the art knowsthat binding affinity may be enhanced by making amino acid substitutionswithin the CDRs or within the hypervariable loops (Chothia and Lesk, JMal. Biol. 196 (1987), 901-917) which partially overlap with the CDRs asdefined by Kabat. Thus, the present invention also relates to antibodieswherein one or more of the mentioned CDRs comprise one or more,preferably not more than two amino acid substitutions. Preferably, theantibody of the invention comprises in one or both of its immunoglobulinchains two or all three CDRs of the variable regions as set forth for VHregions in SEQ ID NOs: 2, 10, 18, 22, 30, 38, 76, 84 and 92, for VLregions and SEQ ID NOs: 4, 12, 20, 24, 32, 40, 78, 86 and 94 or asindicated in FIG. 1.

The polynucleotide of the invention encoding the above describedantibody may be, e.g., DNA, cDNA, RNA or synthetically produced DNA orRNA or a recombinantly produced chimeric nucleic acid moleculecomprising any of those polynucleotides either alone or in combination.In one embodiment the polynucleotide is a cDNA encoding the variableregion and at least part of the constant domain. In a preferredembodiment a vector comprising the above polynucleotide is provided,optionally in combination with said polynucleotide which encodes thevariable region of the other immunoglobulin chain of said antibody. Suchvectors may comprise further genes such as marker genes which allow forthe selection of said vector in a suitable host cell and under suitableconditions.

Preferably, the polynucleotide of the invention is operatively linked toexpression control sequences allowing expression in prokaryotic oreukaryotic cells. Expression of said polynucleotide comprisestranscription of the polynucleotide into a translatable mRNA. Regulatoryelements ensuring expression in eukaryotic cells, preferably mammaliancells, are well known to those skilled in the art. They usually compriseregulatory sequences ensuring initiation of transcription and optionallypoly-A signals ensuring termination of transcription and stabilizationof the transcript. Additional regulatory elements may includetranscriptional as well as translational enhancers, and/or naturallyassociated or heterologous promoter regions.

In this respect, the person skilled in the art will readily appreciatethat the polynucleotides encoding at least the variable domain of thelight and/or heavy chain may encode the variable domains of bothimmunoglobulin chains or one chain only.

Likewise, said polynucleotides may be under the control of the samepromoter or may be separately controlled for expression. Possibleregulatory elements permitting expression in prokaryotic host cellscomprise, e.g., the PL, lac, trp or tac promoter in E. coli, andexamples for regulatory elements permitting expression in eukaryotichost cells are the AOXI or GALI promoter in yeast or the CMV-, SV40-,RSV-promoter, CMV-enhancer, SV40-enhancer or a globin intron inmammalian and other animal cells.

Beside elements which are responsible for the initiation oftranscription such regulatory elements may also comprise transcriptiontermination signals, such as the SV40-poly-A site or the tk-poly-A site,downstream of the polynucleotide. Furthermore, depending on theexpression system used leader sequences capable of directing thepolypeptide to a cellular compartment or secreting it into the mediummay be added to the coding sequence of the polynucleotide of theinvention and are well known in the art. The leader sequence(s) is (are)assembled in appropriate phase with translation, initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein, or a portion thereof, intothe periplasmic space or extracellular medium. Optionally, theheterologous sequence can encode a fusion protein including a C- orN-terminal identification peptide imparting desired characteristics,e.g., stabilization or simplified purification of expressed recombinantproduct. In this context, suitable expression vectors are known in theart such as Okayama-Berg cDNA expression vector pcDV1 (Pharmacia),pCDM8, pRc/CMV, pcDNA1, pcDNA3 (Invitrogen), or pSPORT1 (GIBCO BRL).

Preferably, the expression control sequences will be eukaryotic promotersystems in vectors capable of transforming or transfecting eukaryotichost cells, but control sequences for prokaryotic hosts may also beused. Once the vector has been incorporated into the appropriate host,the host is maintained under conditions suitable for high levelexpression of the nucleotide sequences, and, as desired, the collectionand purification of the immunoglobulin light chains, heavy chains,light/heavy chain dimers or intact antibodies, binding fragments orother immunoglobulin forms may follow; see, Beychok, Cells ofImmunoglobulin Synthesis, Academic Press, N.Y., (1979).

Furthermore, the present invention relates to vectors, particularlyplasmids, cosmids, viruses and bacteriophages used conventionally ingenetic engineering that comprise a polynucleotide encoding the antigenor preferably a variable domain of an immunoglobulin chain of anantibody of the invention; optionally in combination with apolynucleotide of the invention that encodes the variable domain of theother immunoglobulin chain of the antibody of the invention. Preferably,said vector is an expression vector and/or a gene transfer or targetingvector.

Expression vectors derived from viruses such as retroviruses, vacciniavirus, adeno-associated virus, herpes viruses, or bovine papillomavirus, may be used for delivery of the polynucleotides or vector of theinvention into targeted cell population. Methods which are well known tothose skilled in the art can be used to construct recombinant viralvectors; see, for example, the techniques described in Sambrook,Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory(1989) N.Y. and Ausubel, Current Protocols in Molecular Biology, GreenPublishing Associates and Wiley Interscience, N.Y. (1994).Alternatively, the polynucleotides and vectors of the invention can bereconstituted into liposomes for delivery to target cells. The vectorscontaining the polynucleotides of the invention (e.g., the heavy and/orlight variable domain(s) of the immunoglobulin chains encoding sequencesand expression control sequences) can be transferred into the host cellby well-known methods, which vary depending on the type of cellularhost. For example, calcium chloride transfection is commonly utilizedfor prokaryotic cells, whereas calcium phosphate treatment orelectroporation may be used for the transformation of other cellularhosts; see Sambrook, supra. In respect to the above, the presentinvention furthermore relates to a host cell comprising saidpolynucleotide or vector. Said host cell may be a prokaryotic oreukaryotic cell. The polynucleotide or vector of the invention which ispresent in the host cell may either be integrated into the genome of thehost cell or it may be maintained extrachromosomally. The host cell canbe any prokaryotic or eukaryotic cell, such as a bacterial, insect,fungal, plant, animal or human cell; suitable host cells and methods forproduction of the antibodies of the present invention are described inmore detail in the section “Host cells” below.

Using the above-mentioned host cells it is possible to produce andprepare an antibody of the present invention for, e.g., a pharmaceuticaluse or as a target for therapeutic intervention. Therefore, in oneembodiment, it is also an object of the present invention to provide amethod for preparing an anti-IFN-α antibody or IFN-α binding fragmentthereof, said method comprising

-   (a) culturing the cell as defined hereinabove; and-   (b) isolating said antibody or IFN-α binding fragment thereof from    the culture.

Accordingly, the present invention relates to recombinant antibody orIFN-α binding fragment thereof immunoglobulin chain(s) thereof encodedby the polynucleotide of the present invention or obtainable by theabove-mentioned method for preparing an anti-IFN-α antibody orimmunoglobulin chain(s) thereof Means and methods for the recombinantproduction of antibodies and mimics thereof as well as methods ofscreening for competing binding molecules, which may or may not beantibodies, are known in the art. However, as described herein, inparticular with respect to therapeutic applications in human theantibody of the present invention is a recombinant human antibody in thesense that application of said antibody is substantially free of animmune response directed against such antibody otherwise observed forchimeric and even humanized antibodies.

The binding molecules, antibodies or fragments thereof may be directlyused as a therapeutic. However, in one embodiment the antibody orantigen-binding fragment which is provided by the present invention, isdetectably labeled or attached to a drug, preferably wherein thedetectable label is selected from the group consisting of an enzyme, aradioisotope, a fluorophore, a peptide and a heavy metal. Labeledantibodies or antigen-binding fragments of the present invention may beused to detect specific targets in vivo or in vitro including“immunochemistry/immunolabelling” like assays in vitro. In vivo they maybe used in a manner similar to nuclear medicine imaging techniques todetect tissues, cells, or other material expressing the antigen ofinterest. Labels, their use in diagnostics and their coupling to thebinding molecules of the present invention are described in more detailin section “labels and diagnostics” further below.

The antibodies of the present invention are isolated from animals orhumans affected by an autoimmune disorder. On the other hand, IFN-αspecific antibodies identified in the present invention may be involvedin severely impairing the immune system of the affected individual,which is associated with, e.g., symptoms observed in APECED patients.Therefore, it is a further aspect of the present invention, toextinguish or at least relieve the pathological reactions of subjectssuffering from autoimmune disorders by providing means and measures tominimize the number of auto-antibodies and/or their effects in adiseased human patient or animal Thus, in one embodiment the presentinvention also relates to a peptide or peptide-based compound comprisingan epitope specifically recognized by an autoantibody of the presentinvention. A similar effect as by application of competitive antigens,sequestering and preventing thereby the binding of the autoantibodies totheir respective targets may be obtained by anti-idiotypic antibodies,as described in detail further below. Therefore, in one embodiment thepresent invention also provides an anti-idiotypic antibody of anautoantibody of the present invention.

As already indicated above, the present invention also relates to theanti-idiotypic antibody or the peptide or peptide-based compound of thepresent invention for use in the treatment of a disorder as definedabove, i.e. a disorder associated with a disrupted or deregulatedgenesis of self-tolerance. These isolated antibodies or fragmentsthereof of the present invention can be used as immunogenes to generatea panel of monoclonal anti-idiotypes. For suitable methods for thegeneration of anti-idiotypic antibodies see Raychadhuri et al., JImmunol. 137 (1986), 1743 and for T-cells see Ertl et al., J Exp. Med.159 (1985), 1776. The anti-idiotypic antibodies will be characterizedwith respect to the expression of internal image and non-internal imageidiotypes using standard assays routinely practiced in the art asdescribed in detail by Raychaudhuri et al., J Immunol. 137 (1986), 1743.If an anti-idiotypic antibody structurally mimics the antigen of theantibody it is binding to or bound by, it is called the “internal image”of the antigen.

Methods of providing molecules which mimic an idiotype of an autoimmunedisease-associated auto-antibody (autoantibodies) are described in theart; see, e.g., international application WO03/099868, the disclosurecontent of which incorporated herein by reference. For example, suchmethod may comprise the following steps: (a) providing autoantibodies inaccordance with the method of the present invention; (b) binding theautoantibodies to a solid phase to form an affinity matrix; (c)contacting pooled plasma or B cells comprising immunoglobulins with theaffinity matrix followed by removal of unbound plasma components; (d)eluting bound immunoglobulins, being anti-Idiotypic antibodies (anti-Id)to autoantibodies, from the matrix; (e) providing a molecular librarycomprising a plurality of molecule members; and (f) contacting theanti-Id with the molecular library and isolating those bound moleculeswhich are bound by the anti-Id, the bound molecules being moleculeswhich mimic an idiotype of autoantibodies. A method of isolatingidiotypic autoantibodies in disclosed in international applicationWO2010/136196, the disclosure content of which incorporated herein byreference, which describes immunoglobulin preparations containingnatural polyclonal IgG-reactive antibodies (Abs) isolated from normalhuman serum (NHS), for the treatment of autoimmune diseases and immunesystem disorders. The IgG-reactive Abs potently neutralizedisease-associated or pathogenic autoantibodies present in sera ofpatients suffering from autoimmune diseases, by binding to theirantigenic determinants located either within or near (e.g. overlappingwith) the antigen combining sites.

The present invention also relates to compositions comprising any one ofthe aforementioned anti-IFN-α antibodies and/or IFN-α binding fragments,the polynucleotide, the vector, the cell, the peptide or peptide-basedcompound of the present invention and/or a cocktail of antibodies orIFN-α binding fragments thereof which in combination display thefeatures of an antibody or IFN-α binding fragment thereof of the presentinvention. In addition or alternatively in one embodiment thecomposition or the kit of the present invention comprises theanti-idiotypic antibody of the present invention. In one embodiment thecomposition is a pharmaceutical composition and further comprises apharmaceutically acceptable carrier. Pharmaceutically acceptablecarriers, administration routes and dosage regimen can be taken fromcorresponding literature known to the person skilled in the art and aredescribed as well in more detail in sections “Pharmaceutical carriers”and “Dosage regimen” further below.

Besides biochemical and cell based in vitro assays therapeutic utilityof the antibodies of the present invention can be validated inappropriate animal models such as for RA, psoriasis, SLE or TIDM; seethe Examples below, e.g., Example 4.

In one embodiment the pharmaceutical composition further comprises anadditional agent useful for treating an inflammation or an autoimmunedisorder, preferably wherein said agent is selected from the groupconsisting of Non-Steroidal Antiinflammatory Drugs (NSAIDs),Corticosteroids, Anti-Histamines and combinations thereof. In additionor alternatively, in a further embodiment the pharmaceutical compositionfurther comprises an additional agent useful for treating aninflammation related disease, selected from the group consisting ofimmunosuppressive and anti-inflammatory or “anti-rheumatic” drugs.

In another embodiment, the composition is a diagnostic composition orkit and further comprises reagents conventionally used in immuno- ornucleic acid based diagnostic methods.

Furthermore, the present invention relates to any one of theaforementioned anti-IFN-a antibodies and IFN-α binding fragment thereof,or the composition as defined hereinabove for use in a method of:

-   (a) treating or preventing the progression of an immune mediated or    autoimmune disease or condition;-   (b) amelioration of symptoms associated with an immune mediated or    autoimmune disease or condition; and/or-   (c) diagnosing or screening a subject for the presence or for    determining a subject's risk for developing an immune mediated or    autoimmune disease or condition; wherein the disease or condition    associated with the expression of IFN-α and/or IFN-ω in a patient

In this respect several application routes may be used. In oneembodiment of the present invention the aforementioned antibody orantigen-binding fragment, the anti-idiotypic antibody or peptide orpeptide-based compound and/or a cocktail of antibodies which incombination display the features of an antibody of the present inventionis provided, which is designed to be administered intravenously,intramuscularly, subcutaneously, intraperitoneally, intranasally,parenterally or as an aerosol. As indicated above, due to their bindingspecificity, the molecules of the present invention such as antibodiesand fragments thereof may preferably be used in the above defined methodof treatment, amelioration, diagnosing and/or screening of an immunemediated or autoimmune disorder or condition. For example, elevatedIFN-α activity has been frequently detected in the sera of patients withSLE (R6nnblom et al., Sem. Immun. 23 (2011), 113-121). A specificexpression pattern of interferon-dependent genes (termed the “interferonsignature”) is further displayed in the leukocytes of patients withvarious autoimmune disorders such as Sj6gren's syndrome,Dermatomyositis, Multiple Sclerosis (MS), Psoriasis, type 1 or insulindependent diabetes mellitus (T1DM or IDDM) and a fraction of RA patients(see, e.g., Higgs et al., Eur Muse Rev (2012), 22-28). In addition,development of inflammatory arthritis, MS and T1DM has been repeatedlyobserved during IFN-α therapy indicating that IFN-α at least promotesthose diseases (Crow M K., Arthritis Res Ther. (2010), Suppl 1:S5).Further data suggest an involvement of IFN-α in myositis, systemicscleroderma, chronic psoriasis (Higgs et al., Eur Muse Rev (2012),22-28; Bissonnette et al., J Am Acad Dermatol (2009), 427-436; GreenbergS A, Arth Res Ther (2010): S4;) and autoimmune thyroiditis (Prummel andLaurberg, Thyroid (2003), 547-551). Therefore, in one embodiment theantibody or IFN-α binding fragment thereof or the composition as definedhereinabove for use in the above-mentioned method is provided, whereinsaid disease is an autoimmune disease, preferably selected from thegroup consisting of Systemic lupus erythematosus (SLE), cutaneous lupuserythematosus (CLE), discoid lupus erythematosus (DLE), type 1 diabetesmellitus (T1DM) Sj6gren's syndrome, dermatomyositis, psoriasis,autoimmune thyroiditis, rheumatoid arthritis, spondyloarthritis,scleroderma and different cancer forms including leukemia, such asbreast and ovarian cancer and childhood lymphoblastic leukemia (ALL;Einav et al., Oncogene 24 (2005), 6367-6375). Preliminary resultssuggested a difference in neutralizing activity of antibodies obtainedfrom APS1-patients suffering or not suffering in addition from T1DM(FIG. 4). However, a more detailed analysis of the patient's serarevealed that the neutralizing activity of individual anti-IFN-αantibodies in both kind of patients may be substantially the same, whilethe antibody titers and total level of IFN neutralizing activitysignificantly differ with a very low titer in T1DM patients; see FIGS.31 and 32. Accordingly, in one embodiment the present invention alsorelates to the IFN-α and IFN-α/m binding molecules described herein aswell as other IFN-a binding molecules for use in the treatment of T1DMin a patient, from whom a sample of sera has been determined to displaya lower neutralizing activity against cytokines of at least one IFN-αsubtype compared to a control sample, i.e. sera from a healthy subjector from a TD1M patient which is free of disease symptoms.

Due to the multitude of molecules suitable in treatment of, e.g.,disorders associated with inflammation presented herein, the presentinvention also relates to methods of treatment, diagnosing and/orprognosticate the probable course and outcome of such disorders,preferably wherein the immune mediated or autoimmune disease orcondition is associated with the expression of IFN-α and/or IFN-ω to theuse of the molecules of the present invention. In one embodiment amethod for treating of such a disorder is provided, which methodcomprises administering to a subject in need thereof a therapeuticallyeffective amount of the aforementioned antibody or antigen-bindingfragment, the cocktail of antibodies which in combination display thefeatures of an antibody of the present invention, the anti-idiotypicantibody or the peptide or peptide-based compound.

Furthermore, in one embodiment the present invention relates to a methodof treating an immune mediated or autoimmune disease or conditionassociated with the expression of IFN-α and/or IFN-ω comprisingadministering to a subject a therapeutically effective amount of aligand binding molecule comprising:

-   (i) at least one CDR of the anti-IFN-α antibodies and IFN-α binding    fragment of the present invention; or-   (ii) at least one anti-idiotypic antibody and/or peptide or    peptide-based compound as defined hereinabove.

Treatment methods based on the use of only one monoclonal antibodyspecific for an epitope of a particular antigen, which is related orcausing a disease may suffer from several shortcomings. For example,difficulties and probably inefficiency of treatment may stem from themultiplicity of the pathogenic mechanisms causing a specific disorderrequiring targeting of several antigens simultaneously. Furthermore, theinherent diversity of the patient population has to be taken intoaccount concerning, e.g., polymorphism, heterogeneity of glycosylationor slight denaturation of a given antigen, either in different or in onepatient which may lead to a decreased binding efficiency of themonoclonal antibody used at least. Some of these shortcomings may becircumvented by, e.g., pretreatment screenings to determine whether theantigen is immunologically relevant to the patients intended to betreated and whether there are any epitope changes in the particularpatients. However, such screenings are often omitted either due totreatment urgency or to cost restraints. Therefore, the presentinvention further relates to methods based on the application of morethan one type of a binding molecule at once to a patient, i.e. to theapplication of a cocktail of binding molecules. These binding moleculesmay specifically bind to one IFN-α subtype at different epitopes and/orof an epitope of IFN-ω, each of the binding molecules applied may bindspecifically another IFN-α subtype or several binding molecules are usedbinding to several epitopes of more than one IFN-α subtype and/or IFN-ω.In case the binding molecules of the present invention are directed(bind specifically) towards one IFN-α subtype as antigen, their bindingspecificity is directed towards distinct epitopes of said antigen. Theuse of such cocktails is in particular envisaged for the treatment ofpatients suffering from autoimmune disorders such as APSI, who in viewof the presence of autoantibodies against about 3000 endogenous antigensare often not amenable to monotherapy with one particular antibody. Insuch cases, combination therapy with two or more monoclonal antibodiesand/or peptides and peptide-based compounds of the present inventionwith the same or different antigen specificity are expected to achieveat least some relief of the symptoms.

Therefore, in one embodiment a further method of treating a disorder isprovided comprising administering to a subject a therapeuticallyeffective amount of a cocktail consisting essentially of at least two,three, four, five and more components selected from the groupsconsisting of: an anti-IFN-α a antibody and IFN-α binding fragment ofthe present invention specifically binding the IFN-α subtypes and/orIFN-ω as defined hereinabove; and/or an anti-idiotypic antibody of thepresent invention, and/or from a peptide or peptide-based compound ofthe present invention, which peptide or peptide-based compound comprisesan epitope specifically recognized by an anti-IFN-α antibodies and IFN-αbinding fragment of the present invention.

The present invention naturally extents also to diagnostic andprognostic methods directed towards diagnosing immune mediated orautoimmune conditions and disorders associated with expression of one ormore subtypes of IFN-α and/or IFN-ω and/or prognosis of the developmentof the disease, i.e. its progression, response to treatment or recovery.Therefore, in one embodiment the present invention relates to a methodfor diagnosing an immune mediated or autoimmune disease or condition ina subject associated with the expression of IFN-α comprising contactinga biological sample of the subject with anti-IFN-α antibody and IFN-abinding fragment of the present invention, and detecting the presence ofIFN-α and/or IFN-m. Furthermore, in one embodiment the present inventionrelates to a method of detecting or determining IFN-α and/or IFN-ω in anisolated biological sample comprising admixing the sample with ananti-IFN-α antibody of the present invention, allowing the antibody toform a complex with any IFN-α subtype and/or IFN-ω present in themixture, and detecting the complex present in the mixture.

As already mentioned above, in one embodiment the present inventionrelates to a kit for the diagnosis of an immune mediated or autoimmunedisease or condition associated with the expression of IFN-α and/orIFN-ω, said kit comprising the aforementioned anti-IFN-a antibody andIFN-α binding fragment, the anti-idiotypic antibody or the peptide orpeptide-based compound, the polynucleotide, the vector or the cell,optionally with reagents and/or instructions for use. Associated withthe kits of the present invention, e.g., within a container comprisingthe kit can be a notice in the form prescribed by a governmental agencyregulating the manufacture, use or sale of pharmaceuticals or biologicalproducts, which notice reflects approval by the agency of manufacture,use or sale for human administration. In addition or alternatively thekit comprises reagents and/or instructions for use in appropriatediagnostic assays. The compositions, i.e. kits of the present inventionare of course particularly suitable for the diagnosis, prevention andtreatment of a disorder which is accompanied with the expression ofIFN-α, in particular applicable for the treatment of diseases asmentioned above. In a particularly preferred embodiment the disorder isassociated with expression of one or more of IFN-α subtypes and/orIFN-ω.

In another embodiment the present invention relates to a diagnosticcomposition comprising any one of the above described binding molecules,antibodies, antigen-binding fragments, peptides or peptide-basedcompounds, polynucleotides, vectors or cells of the invention andoptionally suitable means for detection such as reagents conventionallyused in immune- or nucleic acid based diagnostic methods. The antibodiesof the invention are, for example, suited for use in immunoassays inwhich they can be utilized in liquid phase or bound to a solid phasecarrier. Examples of immunoassays which can utilize the antibody of theinvention are competitive and non-competitive immunoassays in either adirect or indirect format. Examples of such immunoassays are theradioimmunoassay (RIA), the sandwich (immunometric assay), flowcytometry and the Western blot assay. The antigens and antibodies of theinvention can be bound to many different carriers and used to isolatecells specifically bound thereto. Examples of well-known carriersinclude glass, polystyrene, polyvinyl chloride, polypropylene,polyethylene, polycarbonate, dextran, nylon, amyloses, natural andmodified celluloses, poly-acrylamides, agaroses, and magnetite. Thenature of the carrier can be either soluble or insoluble for thepurposes of the invention. There are many different labels and methodsof labeling known to those of ordinary skill in the art. Examples of thetypes of labels which can be used in the present invention includeenzymes, radioisotopes, colloidal metals, fluorescent compounds,chemiluminescent compounds, and bioluminescent compounds; see also theembodiments discussed hereinabove. In this context, the presentinvention also relates to means specifically designed for this purpose.For example, a protein- or antibody-based array may be used, which isfor example loaded with either antigens derived from one or more IFN-αsubtypes and containing the disease-associated antigen in order todetect autoantibodies which may be present in patients suffering from anautoimmune diseases, in particular SLE or APECED/APS 1, or withantibodies or equivalent antigen-binding molecules of the presentinvention which specifically recognize any one of thoseinflammation-associated antigens. Design of microarray immunoassays issummarized in Kusnezow et al., Mal. Cell Proteomics 5 (2006), 1681-1696.Accordingly, the present invention also relates to microarrays loadedwith binding molecules, in particular anti-IFN-α antibody and IFN-αbinding fragment of the present invention or antigens identified inaccordance with the present invention.

Definitions and Embodiments

Unless otherwise stated, a term and an embodiment as used herein isgiven the definition as provided and used in international applicationWO2013/098419 and international application WO2013/098420.Supplementary, a common term as used herein is given the definition asprovided in the Oxford Dictionary of Biochemistry and Molecular Biology,Oxford University Press, 1997, revised 2000 and reprinted 2003, ISBN 019 850673 2.

It is to be noted that the term “a” or “an” entity refers to one or moreof that entity; for example, “an antibody,” is understood to representone or more antibodies. As such, the terms “a” (or “an”), “one or more,”and “at least one” can be used interchangeably herein.

The term “neutralizing” and “neutralizing antibody”, respectively, isused as common in the art in that an antibody is meant that reduces orabolishes at least some biological activity of an antigen or of a livingmicroorganism. For example, a subtype-specific anti-IFN-α antibody ofthe present invention is a neutralizing antibody, if, in adequateamounts, it abolishes or reduces the activity of the respective IFN-αsubtype(s) for example in an assay as described in the Examples.Neutralization is commonly defined by 50% inhibitory concentrations (IC50) and can be statistically assessed based on the area under theneutralization titration curves (AUC). IC 50 values of exemplaryanti-IFN-α antibodies of the present invention are described and shownherein, e.g., in FIGS. 8-10 and in Table 4.

Central and Peripheral Tolerance

Central and peripheral tolerance are described in more detail in therespective chapter of the international application WO2013/098419 onpages 62-63, the disclosure content of which is incorporated herein byreference.

Peptides and Polypeptides:

The term “peptide” is understood to include the terms “polypeptide” and“protein” (which, at times, may be used interchangeably herein) and anyamino acid sequence such as those of the heavy and light chain variableregion as well as constant region of the present invention within itsmeaning. Similarly, fragments of proteins and polypeptides are alsocontemplated and may be referred to herein as “peptides”. Nevertheless,the term “peptide” preferably denotes an amino acid polymer including atleast 5 contiguous amino acids, preferably at least 10 contiguous aminoacids, more preferably at least 15 contiguous amino acids, still morepreferably at least 20 contiguous amino acids, and particularlypreferred at least 25 contiguous amino acids. In addition, the peptidein accordance with present invention typically has no more than 100contiguous amino acids, preferably less than 80 contiguous amino acidsand more preferably less than 50 contiguous amino acids.

As used herein, the term “polypeptide” is intended to encompass asingular “polypeptide” as well as plural “polypeptides” such asantibodies of the present invention, and refers to a molecule composedof monomers (amino acids) linearly linked by amide bonds (also known aspeptide bonds). The term “polypeptide” refers to any chain or chains oftwo or more amino acids, and does not refer to a specific length of theproduct. Thus. “peptides,” “dipeptides,” “tripeptides, “oligopeptides,”“protein,” “amino acid chain,” or any other term used to refer to achain or chains of two or more amino acids, are included within thedefinition of “polypeptide,” and the term “polypeptide” may be usedinstead of, or interchangeably with any of these terms.

The term “polypeptide” is also intended to refer to the products ofpost-expression modifications of the polypeptide, including withoutlimitation glycosylation, acetylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, or modification by non-naturally occurring amino acids. Apolypeptide may be derived from a natural biological source or producedby recombinant technology, but is not necessarily translated from adesignated nucleic acid sequence. It may be generated in any manner,including by chemical synthesis.

A polypeptide of the invention may be of a size of about 3 or more, 5 ormore, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 ormore, 200 or more, 500 or more, 1,000 or more, or 2,000 or more aminoacids. Nevertheless, the term “polypeptide” preferably denotes an aminoacid polymer including at least 100 amino acids. Polypeptides may have adefined three-dimensional structure, although they do not necessarilyhave such structure. Polypeptides with a defined three-dimensionalstructure are referred to as folded, and polypeptides which do notpossess a defined three-dimensional structure, but rather can adopt alarge number of different conformations, and are referred to asunfolded. As used herein, the term glycoprotein refers to a proteincoupled to at least one carbohydrate moiety that is attached to theprotein via an oxygen-containing or a nitrogen-containing side chain ofan amino acid residue, e.g., a serine residue or an asparagine residue.

By an “isolated” polypeptide or a fragment, variant, or derivativethereof is intended a polypeptide that is not in its natural milieu. Noparticular level of purification is required. For example, an isolatedpolypeptide can be removed from its native or natural environment.Recombinantly produced polypeptides and proteins expressed in host cellsare considered isolated for purposed of the invention, as are native orrecombinant polypeptides which have been separated, fractionated, orpartially or substantially purified by any suitable technique.

“Recombinant peptides, polypeptides or proteins” refer to peptides,polypeptides or proteins produced by recombinant DNA techniques, i.e.produced from cells, microbial or mammalian, transformed by an exogenousrecombinant DNA expression construct encoding the fusion proteinincluding the desired peptide. Proteins or peptides expressed in mostbacterial cultures will typically be free of glycan. Proteins orpolypeptides expressed in yeast may have a glycosylation patterndifferent from that expressed in mammalian cells.

Also included as polypeptides of the present invention are fragments,derivatives, analogs and variants of the foregoing polypeptides and anycombination thereof. The terms “fragment,” “variant,” “derivative” and“analog” include peptides and polypeptides having an amino acid sequencesufficiently similar to the amino acid sequence of the natural peptide.The term “sufficiently similar” means a first amino acid sequence thatcontains a sufficient or minimum number of identical or equivalent aminoacid residues relative to a second amino acid sequence such that thefirst and second amino acid sequences have a common structural domainand/or common functional activity. For example, amino acid sequencesthat comprise a common structural domain that is at least about 45%, atleast about 50%, at least about 55%, at least about 60%, at least about65%, at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 91%, at least about92%, at least about 93%, at least about 94%, at least about 95%, atleast about 96%, at least about 97%, at least about 98%, at least about99%, or at least about 100%, identical are defined herein assufficiently similar. Preferably, variants will be sufficiently similarto the amino acid sequence of the preferred peptides of the presentinvention, in particular to antibodies or antibody fragments, or tosynthetic peptide or peptide-based compound comprising epitopesrecognized by the antibodies of the present invention or fragments,variants, derivatives or analogs of either of them. Such variantsgenerally retain the functional activity of the peptides of the presentinvention, i.e. are bound by the antibodies of the present invention.Variants include peptides that differ in amino acid sequence from thenative and wt peptide, respectively, by way of one or more amino aciddeletion(s), addition(s), and/or substitution(s). These may be naturallyoccurring variants as well as artificially designed ones.

The terms “fragment,” “variant,” “derivative” and “analog” whenreferring to antibodies or antibody polypeptides of the presentinvention include any polypeptides which retain at least some of theantigen-binding properties of the corresponding native binding molecule,antibody, or polypeptide. Fragments of polypeptides of the presentinvention include proteolytic fragments, as well as deletion fragments,in addition to specific antibody fragments discussed elsewhere herein.Variants of antibodies and antibody polypeptides of the presentinvention include fragments as described above, and also polypeptideswith altered amino acid sequences due to amino acid substitutions,deletions, or insertions. Variants may occur naturally or benon-naturally occurring. Non-naturally occurring variants may beproduced using art-known mutagenesis techniques. Variant polypeptidesmay comprise conservative or non-conservative amino acid substitutions,deletions or additions. Derivatives of binding molecules of the presentinvention, e.g., antibodies and antibody polypeptides of the presentinvention, are polypeptides which have been altered so as to exhibitadditional features not found on the native polypeptide. Examplesinclude fusion proteins. Variant polypeptides may also be referred toherein as “polypeptide analogs”. As used herein a “derivative” of abinding molecule or fragment thereof, an antibody, or an antibodypolypeptide refers to a subject polypeptide having one or more residueschemically derivatized by reaction of a functional side group. Alsoincluded as “derivatives” are peptides which contain one or morenaturally occurring amino acid derivatives of the twenty standard aminoacids. For example, 4-hydroxyproline may be substituted for proline;5-hydroxylysine may be substituted for lysine; 3-methylhistidine may besubstituted for histidine; homoserine may be substituted for serine; andornithine may be substituted for lysine.

Anti-Idiotypic Antibodies:

The term “anti-idiotypic antibodies” when referring to antibodies orother binding molecules includes molecules which bind to the uniqueantigenic peptide sequence located on an antibody's variable region nearor at the antigen binding site, inhibiting by this a specific immuneresponse by otherwise caused by the given auto-antibody. In an analogousmanner synthetic peptide or peptide-based compound comprising an epitopespecifically recognized by an antibody of the present invention may beused.

Anti-idiotypic antibodies may be obtained in a similar fashion as otherantibodies. The particular anti-idiotypic antibody is detected by anysort of cross-linking, either by agglutination (in turbidimetric ornephelometric assays), precipitation (radial immunodiffusion), orsandwich immunoassays such as ELISAs. U.S. patent application No.20020142356 provides a method for obtaining anti-idiotypic monoclonalantibody populations directed to an antibody that is specific for ahigh-concentration, high-molecular-weight target antigen wherein saidanti-idiotypic antibody populations have a wide range of bindingaffinities for the selected antibody specific to said target antigen andwherein a subset of said anti-idiotypic antibody populations can beselected having the required affinity for a particular application.

U.S. patent application No. 20020142356 describes a competitiveimmunoassay of an antigen using an antibody as coat and ananti-idiotypic antibody as detection or vice-versa. Other referencesdisclosing use of an anti-idiotypic antibody as a surrogate antigeninclude Losman et al., Cancer Research, 55 (1995) (23 suppl.S):S5978-S5982; Becker et al., J of Immunol. Methods 192 (1996), 73-85;Baral et al., International J of Cancer, 92 (2001), 88-95; and Kohen etal., Food and Agriculture Immunology, 12 (2000), 193-201. Use ofanti-idiotypic antibodies in treatment of diseases or against parasitesis known in the art; see, e.g., in Sacks et al., J Exper. Medicine, 155(1982), 1108-1119.

Determination of Similarity and/or Identity of Molecules:

“Similarity” between two peptides is determined by comparing the aminoacid sequence of one peptide to the sequence of a second peptide. Anamino acid of one peptide is similar to the corresponding amino acid ofa second peptide if it is identical or a conservative amino acidsubstitution. Conservative substitutions include those described inDayhoff, M. O., ed., The Atlas of Protein Sequence and Structure 5,National Biomedical Research Foundation, Washington, D.C. (1978), and inArgos, EMBO J 8 (1989), 779-785. For example, amino acids belonging toone of the following groups represent conservative changes orsubstitutions: -Ala, Pro, Gly, Gln, Asn, Ser, Thr; -Cys, Ser, Tyr, Thr;-Val, Ile, Leu, Met, Ala, Phe; -Lys, Arg, His;-Phe, Tyr, Trp, His; and-Asp, Glu.

The determination of percent identity or similarity between twosequences is preferably accomplished using the mathematical algorithm ofKarlin and Altschul (1993) Proc. Natl. Acad. Sci USA 90: 5873-5877. Suchan algorithm is incorporated into the BLASTn and BLASTp programs ofAltschul et al. (1990) J Mal. Biol. 215: 403-410 available at NCBI(http://www.ncbi.nlm.nih.gov/blast/Blast.cge).

The determination of percent identity or similarity is performed withthe standard parameters of the BLASTn and BLASTp programs. BLASTpolynucleotide searches are performed with the BLASTn program. For thegeneral parameters, the “Max Target Sequences” box may be set to 100,the “Short queries” box may be ticked, the “Expect threshold” box may beset to 10 and the “Word Size” box may be set to 28. For the scoringparameters the “Match/mismatch Scores” may be set to 1,-2 and the “GapCosts” box may be set to linear. For the Filters and Masking parameters,the “Low complexity regions” box may not be ticked, the“Species-specific repeats” box may not be ticked, the “Mask for lookuptable only” box may be ticked, and the “Mask lower case letters” box maynot be ticked.

BLAST protein searches are performed with the BLASTp program. For thegeneral parameters, the “Max Target Sequences” box may be set to 100,the “Short queries” box may be ticked, the “Expect threshold” box may beset to 10 and the “Word Size” box may be set to “3”. For the scoringparameters the “Matrix” box may be set to “BLOSUM62”, the “Gap Costs”Box may be set to “Existence: 11 Extension: 1”, the “Compositionaladjustments” box may be set to “Conditional compositional score matrixadjustment”. For the Filters and Masking parameters the “Low complexityregions” box may not be ticked, the “Mask for lookup table only” box maynot be ticked and the “Mask lower case letters” box may not be ticked.

Polynucleotides:

The term “polynucleotide” is intended to encompass a singular nucleicacid as well as plural nucleic acids, and refers to an isolated nucleicacid molecule or construct, e.g., messenger RNA (mRNA) or plasmid DNA(pDNA). A polynucleotide may comprise a conventional phosphodiester bondor a non-conventional bond (e.g., an amide bond, such as found inpeptide nucleic acids (PNA)). The term “nucleic acid” refers to any oneor more nucleic acid segments, e.g., DNA or RNA fragments, present in apolynucleotide. By “isolated” nucleic acid or polynucleotide is intendeda nucleic acid molecule, DNA or RNA, which has been removed from itsnative environment. For example, a recombinant polynucleotide encodingan antibody contained in a vector is considered isolated for thepurposes of the present invention. Further examples of an isolatedpolynucleotide include recombinant polynucleotides maintained inheterologous host cells or purified (partially or substantially)polynucleotides in solution. Isolated RNA molecules include in vivo orin vitro RNA transcripts of polynucleotides of the present invention.Isolated polynucleotides or nucleic acids according to the presentinvention further include such molecules produced synthetically. Inaddition, polynucleotide or a nucleic acid may be or may include aregulatory element such as a promoter, ribosome binding site, or atranscription terminator.

As used herein, a “coding region” is a portion of nucleic acid whichconsists of codons translated into amino acids. Although a “stop codon”(TAG, TGA, or TAA) is not translated into an amino acid, it may beconsidered to be part of a coding region, but any flanking sequences,for example promoters, ribosome binding sites, transcriptionalterminators, introns, and the like, are not part of a coding region. Twoor more coding regions of the present invention can be present in asingle polynucleotide construct, e.g., on a single vector, or inseparate polynucleotide constructs, e.g., on separate (different)vectors. Furthermore, any vector may contain a single coding region, ormay comprise two or more coding regions, e.g., a single vector mayseparately encode an immunoglobulin heavy chain variable region and animmunoglobulin light chain variable region. In addition, a vector,polynucleotide, or nucleic acid of the invention may encode heterologouscoding regions, either fused or not fused to a nucleic acid encoding abinding molecule, an antibody, or fragment, variant, or derivativethereof. Heterologous coding regions include without limitationspecialized elements or motifs, such as a secretory signal peptide or aheterologous functional domain. In certain embodiments, thepolynucleotide or nucleic acid is DNA. In the case of DNA, apolynucleotide comprising a nucleic acid which encodes a polypeptidenormally may include a promoter and/or other transcription ortranslation control elements operably associated with one or more codingregions. An operable association is when a coding region for a geneproduct, e.g., a polypeptide, is associated with one or more regulatorysequences in such a way as to place expression of the gene product underthe influence or control of the regulatory sequence(s). Two DNAfragments (such as a polypeptide coding region and a promoter associatedtherewith) are “operably associated” or “operably linked” if inductionof promoter function results in the transcription of mRNA encoding thedesired gene product and if the nature of the linkage between the twoDNA fragments does not interfere with the ability of the expressionregulatory sequences to direct the expression of the gene product orinterfere with the ability of the DNA template to be transcribed. Thus,a promoter region would be operably associated with a nucleic acidencoding a polypeptide if the promoter was capable of effectingtranscription of that nucleic acid. The promoter may be a cell-specificpromoter that directs substantial transcription of the DNA only inpredetermined cells. Other transcription control elements, besides apromoter, for example enhancers, operators, repressors, andtranscription termination signals, can be operably associated with thepolynucleotide to direct cell-specific transcription. Suitable promotersand other transcription control regions are disclosed herein.

A variety of transcription control regions are known to those skilled inthe art. These include, without limitation, transcription controlregions which function in vertebrate cells, such as, but not limited to,promoter and enhancer segments from cytomegaloviruses (the immediateearly promoter, in conjunction with intron-A), simian virus 40 (theearly promoter), and retroviruses (such as Rous sarcoma virus). Othertranscription control regions include those derived from vertebrategenes such as actin, heat shock protein, bovine growth hormone andrabbit B-globin, as well as other sequences capable of controlling geneexpression in eukaryotic cells. Additional suitable transcriptioncontrol regions include tissue-specific promoters and enhancers as wellas lymphokine-inducible promoters (e.g., promoters inducible byinterferons or interleukins).

Similarly, a variety of translation control elements are known to thoseof ordinary skill in the art. These include, but are not limited toribosome binding sites, translation initiation and termination codons,and elements derived from picomaviruses (particularly an internalribosome entry site, or IRES, also referred to as a CITE sequence).

In other embodiments, a polynucleotide of the present invention is RNA,for example, in the form of messenger RNA (mRNA), small hairpin RNA(shRNA), small interfering RNA (siRNA) or any other RNA product.

Polynucleotide and nucleic acid coding regions of the present inventionmay be associated with additional coding regions which encode secretoryor signal peptides, which direct the secretion of a polypeptide encodedby a polynucleotide of the present invention. According to the signalhypothesis, proteins secreted by mammalian cells have a signal peptideor secretory leader sequence which is cleaved from the mature proteinonce export of the growing protein chain across the rough endoplasmicreticulum has been initiated. Those of ordinary skill in the art areaware that polypeptides secreted by vertebrate cells generally have asignal peptide fused to the N-terminus of the polypeptide, which iscleaved from the complete or “full-length” polypeptide to produce asecreted or “mature” form of the polypeptide. In certain embodiments,the native signal peptide, e.g., an immunoglobulin heavy chain or lightchain signal peptide is used, or a functional derivative of thatsequence that retains the ability to direct the secretion of thepolypeptide that is operably associated with it. Alternatively, aheterologous mammalian signal peptide, or a functional derivativethereof, may be used. For example, the wild-type leader sequence may besubstituted with the leader sequence of human tissue plasminogenactivator (TPA) or mouse B-glucuronidase. However, intracellularproduction of the polypeptides, in particular of the immunoglobulins andfragments thereof of the present invention is also possible.

Expression:

The term “expression” as used herein refers to a process by which a geneproduces a biochemical, for example, an RNA or polypeptide. The processincludes any manifestation of the functional presence of the gene withinthe cell including, without limitation, gene knockdown as well as bothtransient expression and stable expression. It includes withoutlimitation transcription of the gene into messenger RNA (mRNA), transferRNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA) orany other RNA product, and the translation of such mRNA intopolypeptide(s). If the final desired product is a biochemical,expression includes the creation of that biochemical and any precursors.Expression of a gene produces a “gene product.” As used herein, a geneproduct can be either a nucleic acid, e.g., small interfering RNA(siRNA), a messenger RNA produced by transcription of a gene, or apolypeptide which is translated from a transcript. Gene productsdescribed herein further include nucleic acids with post transcriptionalmodifications, e.g., polyadenylation, or polypeptides with posttranslational modifications, e.g., methylation, glycosylation, theaddition of lipids, association with other protein subunits, proteolyticcleavage, and the like.

A variety of expression vector/host systems may be utilized to containand express polynucleotide sequences. These include, but are not limitedto, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems.

To express the peptide, polypeptide or fusion protein (hereinafterreferred to as “product”) in a host cell, a procedure such as thefollowing can be used. A restriction fragment containing a DNA sequencethat encodes said product may be cloned into an appropriate recombinantplasmid containing an origin of replication that functions in the hostcell and an appropriate selectable marker. The plasmid may include apromoter for inducible expression of the product (e.g., pTrc (Amann etal, Gene 69 (1988), 301 315) and pET1 Id (Studier et al., GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990), 60 89). The recombinant plasmid may be introducedinto the host cell by, for example, electroporation and cells containingthe recombinant plasmid may be identified by selection for the marker onthe plasmid. Expression of the product may be induced and detected inthe host cell using an assay specific for the product.

In some embodiments, the DNA that encodes the product/peptide may beoptimized for expression in the host cell. For example, the DNA mayinclude codons for one or more amino acids that are predominant in thehost cell relative to other codons for the same amino acid.

Alternatively, the expression of the product may be performed by invitro synthesis of the protein in cell-free extracts which are alsoparticularly suited for the incorporation of modified or unnatural aminoacids for functional studies; see also infra. The use of in vitrotranslation systems can have advantages over in vivo gene expressionwhen the over-expressed product is toxic to the host cell, when theproduct is insoluble or forms inclusion bodies, or when the proteinundergoes rapid proteolytic degradation by intracellular proteases. Themost frequently used cell-free translation systems consist of extractsfrom rabbit reticulocytes, wheat germ and Escherichia coli. All areprepared as crude extracts containing all the macromolecular components(70S or 80S ribosomes, tRNAs, aminoacyl-tRNA synthetases, initiation,elongation and termination factors, etc.) required for translation ofexogenous RNA. To ensure efficient translation, each extract must besupplemented with amino acids, energy sources (ATP, GTP), energyregenerating systems (creatine phosphate and creatine phosphokinase foreukaryotic systems, and phosphoenol pyruvate and pyruvate kinase for theE. coli lysate), and other co-factors known in the art (Mg² ₊, K+,etc.). Appropriate transcription/translation systems are commerciallyavailable, for example from Promega Corporation, Roche Diagnostics, andAmbion, i.e. Applied Biosystems (Anderson, C. et al., Meth. Enzymol. 101(1983), 635-644; Arduengo, M. et al. (2007), The Role of Cell-FreeRabbit Reticulocyte Expression Systems in Functional Proteomics in,Kudlicki. Katzen and Bennett eds., Cell-Free Expression Vol. 2007.Austin, Tex.: Landes Bioscience, pp. 1-18; Chen and Zubay, Meth.Enzymol. 101 (1983), 674-90; Ezure et al., Biotechnol. Prag. 22 (2006),1570-1577).

Host Cells:

In respect of the present invention, host cell can be any prokaryotic oreukaryotic cell, such as a bacterial, insect, fungal, plant, animal orhuman cell. Preferred fungal cells are, for example, those of the genusSaccharomyces, in particular those of the species S. cerevisiae. Theterm “prokaryotic” is meant to include all bacteria which can betransformed or transfected with a DNA or RNA molecules for theexpression of an antibody of the invention or the correspondingimmunoglobulin chains Prokaryotic hosts may include gram negative aswell as gram positive bacteria such as, for example, E. coli, S.typhimurium, Serratia marcescens and Bacillus subtilis. The term“eukaryotic” is meant to include yeast, higher plant, insect andpreferably mammalian cells, most preferably HEK 293, NSO, CSO and CHOcells. Depending upon the host employed in a recombinant productionprocedure, the antibodies or immunoglobulin chains encoded by thepolynucleotide of the present invention may be glycosylated or may benon-glycosylated. Antibodies of the invention or the correspondingimmunoglobulin chains may also include an initial methionine amino acidresidue. A polynucleotide of the invention can be used to transform ortransfect the host using any of the techniques commonly known to thoseof ordinary skill in the art. Furthermore, methods for preparing fused,operably linked genes and expressing them in, e g., mammalian cells andbacteria are well-known in the art (Sambrook, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., 1989). The genetic constructs and methods described therein can beutilized for expression of the antibody of the invention or thecorresponding immunoglobulin chains in eukaryotic or prokaryotic hosts.In general, expression vectors containing promoter sequences whichfacilitate the efficient transcription of the inserted polynucleotideare used in connection with the host. The expression vector typicallycontains an origin of replication, a promoter, and a terminator, as wellas specific genes which are capable of providing phenotypic selection ofthe transformed cells. Suitable source cells for the DNA sequences andhost cells for immunoglobulin expression and secretion can be obtainedfrom a number of sources, such as the American Type Culture Collection(“Catalogue of Cell Lines and Hybridomas,” Fifth edition (1985)Rockville, Md., U.S.A., which is incorporated herein by reference).Furthermore, transgenic animals, preferably mammals, comprising cells ofthe invention may be used for the large scale production of the antibodyof the invention.

The transformed hosts can be grown in fermentors and cultured accordingto techniques known in the art to achieve optimal cell growth. Onceexpressed, the whole antibodies, their dimers, individual light andheavy chains, or other immunoglobulin forms of the present invention,can be purified according to standard procedures of the art, includingammonium sulfate precipitation, affinity columns, column chromatography,gel electrophoresis and the like; see, Scopes, “Protein Purification”,Springer Verlag, N.Y. (1982). The antibody or its correspondingimmunoglobulin chain(s) of the invention can then be isolated from thegrowth medium, cellular lysates, or cellular membrane fractions. Theisolation and purification of the, e.g., recombinantly expressedantibodies or immunoglobulin chains of the invention may be by anyconventional means such as, for example, preparative chromatographicseparations and immunological separations such as those involving theuse of monoclonal or polyclonal antibodies directed, e.g., against theconstant region of the antibody of the invention. It will be apparent tothose skilled in the art that the antibodies of the invention can befurther coupled to other moieties for, e.g., drug targeting and imagingapplications. Such coupling may be conducted chemically after expressionof the antibody or antigen to site of attachment or the coupling productmay be engineered into the antibody or antigen of the invention at theDNA level. The DNAs are then expressed in a suitable host system, andthe expressed proteins are collected and renatured, if necessary.

Substantially pure immunoglobulins of at least about 90 to 95%homogeneity are preferred, and 98 to 99% or more homogeneity mostpreferred, for pharmaceutical uses. Once purified, partially or tohomogeneity as desired, the antibodies may then be used therapeutically(including extracorporally) or in developing and performing assayprocedures.

The present invention also involves a method for producing cells capableof expressing an antibody of the invention or its correspondingimmunoglobulin chain(s) comprising genetically engineering cells withthe polynucleotide or with the vector of the invention. The cellsobtainable by the method of the invention can be used, for example, totest the interaction of the antibody of the invention with its antigen.

ELISA-Assays:

Enzyme-linked immunosorbent assays (ELISAs) for various antigens includethose based on colorimetry, chemiluminescence, and fluorometry. ELISAshave been successfully applied in the determination of low amounts ofdrugs and other antigenic components in plasma and urine samples,involve no extraction steps, and are simple to carry out. ELISAs for thedetection of antibodies to protein antigens often use direct binding ofshort synthetic peptides to the plastic surface of a microtitre plate.The peptides are, in general, very pure due to their synthetic natureand efficient purification methods using high-performance liquidchromatography. A drawback of short peptides is that they usuallyrepresent linear, but not conformational or discontinuous epitopes. Topresent conformational epitopes, either long peptides or the completenative protein is used. Direct binding of the protein antigens to thehydrophobic polystyrene support of the plate can result in partial ortotal denaturation of the bound protein and loss of conformationalepitopes. Coating the plate with an antibody, which mediates theimmobilization (capture ELISA) of the antigens, can avoid this effect.

However, frequently, overexpressed recombinant proteins are insolubleand require purification under denaturing conditions and renaturation,when antibodies to conformational epitopes are to be analyzed. See, forexample, U.S. patent application No. 20030044870 for a generic ELISAusing recombinant fusion proteins as coat proteins.

Binding Molecules:

A “binding molecule” as used in the context of the present inventionrelates primarily to antibodies, and fragments thereof, but may alsorefer to other non-antibody molecules that bind to the “molecules ofinterest” of the present invention, wherein the molecules of interestare proteins of the class of glycoproteins known as cytokines, inparticular interferons selected from the group of different IFN-αsubtypes. The molecules of interest of the present invention are definedin further detail within the description of the particular embodimentsof the present invention above and below. The binding molecules of thepresent invention include but are not limited to hormones, receptors,ligands, major histocompatibility complex (MHC) molecules, chaperonessuch as heat shock proteins (HSPs) as well as cell-cell adhesionmolecules such as members of the cadherin, intergrin, C-type lectin andimmunoglobulin (Ig) superfamilies. Thus, for the sake of clarity onlyand without restricting the scope of the present invention most of thefollowing embodiments are discussed with respect to antibodies andantibody-like molecules which represent the preferred binding moleculesfor the development of therapeutic and diagnostic agents.

Antibodies:

The terms “antibody” and “immunoglobulin” are used interchangeablyherein. An antibody or immunoglobulin is a molecule binding to amolecule of interest of the present invention as defined hereinabove andbelow, which comprises at least the variable domain of a heavy chain,and normally comprises at least the variable domains of a heavy chainand a light chain. Basic immunoglobulin structures in vertebrate systemsare relatively well understood; see, e.g., Harlow and Lane, Antibodies:A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.1988). The terms “binds” and “recognizes” are used interchangeably inrespect of the binding affinity of the binding molecules of the presentinvention, e.g., antibodies.

Any antibody or immunoglobulin fragment which contains sufficientstructure to specifically bind to the molecules of interest, as definedhereinabove and below, is denoted herein interchangeably as a “bindingmolecule”, “binding fragment” or an “immunospecific fragment.”

Antibodies or antigen-binding fragments, immunospecific fragments,variants, or derivatives thereof of the invention include, but are notlimited to, polyclonal, monoclonal, multispecific, human, humanized,primatized, murinized or chimeric antibodies, single chain antibodies,epitope-binding fragments, e.g., Fab, Fab′ and F(ab′)2, Fd, Fvs,single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs(sdFv), fragments comprising either a VL or VH domain, fragmentsproduced by a Fab expression library, and anti-idiotypic (anti-Id)antibodies (including, e.g., anti-Id antibodies to antibodies disclosedherein). ScFv molecules are known in the art and are described, e.g., inU.S. Pat. No. 5,892,019. In this respect, antigen-binding fragment ofthe antibody can be as well domain antibodies (dAb) also known as singledomain antibodies (sdAB) or nanobodies™ (Ablynx, Gent, Belgium), see,e.g., De Haard et al., J Bacterial. 187 (2005), 4531-4541; Holt et al.,Trends Biotechnol. 21 (2003), 484-490. As will be discussed in moredetail below, the term “immunoglobulin” comprises various broad classesof polypeptides that can be distinguished biochemically. Those skilledin the art will appreciate that heavy chains are classified as gamma,mu, alpha, delta, or epsilon, (y, μ, a, 8, E) with some subclasses amongthem (e.g., y1-y4). It is the nature of this chain that determines the“class” of the antibody as IgG, IgE, IgM, IgD, IgA, and IgY,respectively. The immunoglobulin subclasses (isotypes) e.g., IgG1, IgG2,IgG3, IgG4, IgA1, etc. are well characterized and are known to conferfunctional specialization Immunoglobulin or antibody molecules of theinvention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY),class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, etc.) or subclass ofimmunoglobulin molecule. Modified versions of each of these classes andisotypes are readily discernible to the skilled artisan in view of theinstant disclosure and, Accordingly, are within the scope of the instantinvention. Although all immunoglobulin classes are clearly within thescope of the present invention, the following discussion will generallybe directed to the IgG class of immunoglobulin molecules. With regard toIgG, a standard immunoglobulin molecule comprises two identical lightchain polypeptides of molecular weight approximately 23,000 Daltons, andtwo identical heavy chain polypeptides of molecular weight53,000-70,000. The four chains are typically joined by disulfide bondsin a “Y” configuration wherein the light chains bracket the heavy chainsstarting at the mouth of the “Y” and continuing through the variableregion.

As evident from the classification of the exemplary anti-IFNa antibodiesof the present invention enlisted in Table 1 above, the exemplaryantibodies of the present invention are of the IgG1 class, possiblyimplicating regulatory T-cell responses and/or epithelia in theirinitiation in these AIRE-deficiency states. These findings are confirmedby the classification of corresponding autoantibodies found in theAIRE-deficient mice described by Kamer et al., in Clin. Exp. Immunol.(2012); doi: 10.1111/cei.12024, the disclosure content of which isincorporated herein by reference. Accordingly, in a preferred embodimentof the present invention, the antibodies of the present invention are ofthe IgG type, even more preferred IgG1.

IgG Structure:

Light chains are classified as either kappa or lambda (K, A). Each heavychain class may be bound with either a kappa or lambda light chain. Ingeneral, the light and heavy chains are covalently bonded to each other,and the “tail” portions of the two heavy chains are bonded to each otherby covalent disulfide linkages or non-covalent linkages when theimmunoglobulins are generated either by hybridomas, B cells orgenetically engineered host cells. In the heavy chain, the amino acidsequences run from an N-terminus at the forked ends of the Yconfiguration to the C-terminus at the bottom of each chain.

Both the light and heavy chains are divided into regions of structuraland functional homology. The terms “constant” and “variable” are usedfunctionally. In this regard, it will be appreciated that the variabledomains of both the light (VL) and heavy (VH) chain portions determineantigen recognition and specificity. Conversely, the constant domains ofthe light chain (CL) and the heavy chain (CHI, CH2 or CH3) conferimportant biological properties such as secretion, transplacentalmobility, Fe receptor binding, complement binding, and the like. Byconvention the numbering of the constant region domains increases asthey become more distal from the antigen-binding site or amino-terminusof the antibody. The N-terminal portion is a variable region and at theC-terminal portion is a constant region; the CH3 and CL domains actuallycomprise the carboxy-terminus of the heavy and light chain,respectively.

As indicated above, the variable region allows the antibody toselectively recognize and specifically bind epitopes on antigens. Thatis, the VL domain and VH domain, or subset of the complementaritydetermining regions (CDRs), of an antibody combine to form the variableregion that defines a three dimensional antigen-binding site. Thisquaternary antibody structure forms the antigen-binding site present atthe end of each arm of the Y. More specifically, the antigen-bindingsite is defined by three CDRs on each of the VH and VL chains. Anyantibody or immunoglobulin fragment which contains sufficient structureto specifically bind to a molecule of interest of the present inventionis denoted herein interchangeably as a “binding fragment” or an“immunospecific fragment.”

In naturally occurring antibodies, an antibody comprises sixhypervariable regions, sometimes called “complementarity determiningregions” or “CDRs” present in each antigen-binding domain, which areshort, non-contiguous sequences of amino acids that are specificallypositioned to form the antigen-binding domain as the antibody assumesits three dimensional configuration in an aqueous environment. The“CDRs” are flanked by four relatively conserved “framework” regions or“FRs” which show less inter-molecular variability. The framework regionslargely adopt a-sheet conformation and the CDRs form loops whichconnect, and in some cases form part of, the -sheet structure. Thus,framework regions act to form a scaffold that provides for positioningthe CDRs in correct orientation by inter-chain, non-covalentinteractions. The antigen-binding domain formed by the positioned CDRsdefines a surface complementary to the epitope on the immunoreactiveantigen. This complementary surface promotes the non-covalent binding ofthe antibody to its cognate epitope. The amino acids comprising the CDRsand the framework regions, respectively, can be readily identified forany given heavy or light chain variable region by one of ordinary skillin the art, since they have been precisely defined; see, “Sequences ofProteins of Immunological Interest,” Kabat, E., et al., US. Departmentof Health and Human Services, (1983); and Chothia and Lesk, J Mal. Biol.196 (1987), 901-917, which are incorporated herein by reference in theirentireties.

In the case where there are two or more definitions of a term which isused and/or accepted within the art, the definition of the term as usedherein is intended to include all such meanings unless explicitly statedto the contrary. A specific example is the use of the term“complementarity determining region” (“CDR”) to describe thenon-contiguous antigen combining sites found within the variable regionof both heavy and light chain polypeptides. This particular region hasbeen described by Kabat et al., US. Dept. of Health and Human Services,“Sequences of Proteins of Immunological Interest” (1983) and by Chothiaand Lesk, J Mal. Biol. 196 (1987), 901-917, which are incorporatedherein by reference, where the definitions include overlapping orsubsets of amino acid residues when compared against each other.Nevertheless, application of either definition to refer to a CDR of anantibody or variants thereof is intended to be within the scope of theterm as defined and used herein. The appropriate amino acid residueswhich encompass the CDRs as defined by each of the above citedreferences are set forth below in Table 2 as a comparison. The exactresidue numbers which encompass a particular CDR will vary depending onthe sequence and size of the CDR. Those skilled in the art can routinelydetermine which residues comprise a particular hypervariable region orCDR of the human IgG subtype of antibody given the variable region aminoacid sequence of the antibody.

TABLE 2 CDR Definitions¹ Kabat Chothia VHCDRI 31-35 26-32 VHCDR2 50-6552-58 VHCDR3  95-102  95-102 VL CDRI 24-34 26-32 VLCDR2 50-56 50-52VLCDR3 89-97 91-96 ¹Numbering of all CDR definitions in Table 2 isaccording to the numbering conventions set forth by Kabat et al. (seebelow).

Kabat et al. also defined a numbering system for variable domainsequences that is applicable to any antibody. One of ordinary skill inthe art can unambiguously assign this system of “Kabat numbering” to anyvariable domain sequence, without reliance on any experimental databeyond the sequence itself. As used herein, “Kabat numbering” refers tothe numbering system set forth by Kabat et al., US. Dept. of Health andHuman Services, “Sequence of Proteins of Immunological Interest” (1983).Unless otherwise specified, references to the numbering of specificamino acid residue positions in an antibody or antigen-binding fragment,variant, or derivative thereof of the present invention are according tothe Kabat numbering system, which however is theoretical and may notequally apply to every antibody of the present invention. For example,depending on the position of the first CDR the following CDRs might beshifted in either direction.

In one embodiment, the antibody of the present invention is not IgM or aderivative thereof with a pentavalent structure. Particular, in specificapplications of the present invention, especially therapeutic use, IgMsare less useful than IgG and other bivalent antibodies or correspondingbinding molecules since IgMs due to their pentavalent structure and lackof affinity maturation often show unspecific cross-reactivities and verylow affinity.

In a particularly preferred embodiment, the antibody of the presentinvention is not a polyclonal antibody, i.e. it substantially consistsof one particular antibody species rather than being a mixture obtainedfrom a plasma immunoglobulin sample.

Antibody Fragments, Animalization:

Antibody fragments, including single-chain antibodies, may comprise thevariable region(s) alone or in combination with the entirety or aportion of the following: hinge region, CHI, CH2, and CH3 domains. Alsoincluded in the invention are fragments binding to a molecule ofinterest of the present invention, said fragments comprising anycombination of variable region(s) with a hinge region, CHI, CH2, and CH3domains. Antibodies or immunospecific fragments thereof of the presentinvention equivalent to the monoclonal antibodies isolated in accordancewith the method of the present invention, in particular to the humanmonoclonal antibodies may be from any animal origin including birds andmammals. Preferably, the antibodies are human, murine, donkey, rabbit,goat, guinea pig, camel, llama, horse, or chicken antibodies. In anotherembodiment, the variable region may be condricthoid in origin (e.g.,from sharks).

In a particularly preferred embodiment of the present invention, theantibodies are naturally occurring human monoclonal antibodies orbinding fragments, derivatives and variants thereof cloned from humansubjects, which bind specifically to specific IFN a subtypes of thepresent invention, as defined in detail above and below, e.g., in Table1, the Figures, in particular FIGS. 1 to 4 and in the Examples, e.g., inExamples 2 and 6.

Optionally, the framework region of the human antibody is aligned andadopted in accordance with the pertinent human germ line variable regionsequences in the database; see, e.g., Vbase(http://vbase.mrc-cpe.cam.ac.uk/) hosted by the MRC Centre for ProteinEngineering (Cambridge, UK). For example, amino acids considered topotentially deviate from the true germ line sequence could be due to thePCR primer sequences incorporated during the cloning process. Comparedto artificially generated human-like antibodies such as single chainantibody fragments (scFvs) from a phage displayed antibody library orxenogeneic mice the human monoclonal antibody of the present inventionis characterized by (i) being obtained using the human immune responserather than that of animal surrogates, i.e. the antibody has beengenerated in response to natural IFNα subtypes in their relevantconformation in the human body, (ii) having protected the individualfrom or minimized at least significant the presence of symptoms of adisease, e.g., SLE, and (iii) since the antibody is of human origin therisks of cross-reactivity against self-antigens is minimized. Thus, inaccordance with the present invention the terms “human monoclonalantibody”, “human monoclonal autoantibody”, “human antibody” and thelike are used to denote a IFN-α binding molecule of a particular IFN-αsubtype specificity which is of human origin, i.e. which has beenisolated from a human cell such as a B cell or hybridoma thereof or thecDNA of which has been directly cloned from mRNA of a human cell, forexample a human memory B cell. A human antibody is still considered as“human” even if amino acid substitutions are made in the antibody, e.g.,to improve its binding characteristics.

Antibodies derived from human immunoglobulin libraries or from animalstransgenic for one or more human immunoglobulins and that do not expressendogenous immunoglobulins, as described infra and, for example in, U.S.Pat. No. 5,939,598 by Kucherlapati et al., are denoted human-likeantibodies in order distinguish them from truly human antibodies of thepresent invention.

For example, the paring of heavy and light chains of human-likeantibodies such as synthetic and semi-synthetic antibodies typicallyisolated from phage display do not necessarily reflect the originalparing as it occurred in the original human B cell. Accordingly Fab andscFv fragments obtained from recombinant expression libraries ascommonly used in the prior art can be considered as being artificialwith all possible associated effects on immunogenicity and stability.

In contrast, the present invention provides isolated affinity-maturedantibodies from selected human subjects, which are characterized bytheir therapeutic utility.

Grafted Antibodies {Equivalents)

The invention also relates to grafted antibodies (interchangeablyreferred to as equivalents) containing CDRs derived from the antibodiesof the present invention, such as anti-IFN-a antibodies, respectively.Such grafted CDRs include animalized antibodies, in which CDRs from theantibodies of the present invention have been grafted or in which a CDRcontaining one or more amino acid substitutions is grafted. The CDRs canbe grafted directly into a human framework or an antibody framework fromanimal origin as indicated above. If desired, framework changes can alsobe incorporated by generating framework libraries. The optimization ofCDRs and/or framework sequences can be performed independently andsequentially combined or can be performed simultaneously, as describedin more detail below.

To generate grafted antibodies donor CDRs of the antibodies of thepresent invention are grafted onto an antibody acceptor variable regionframework. Methods for grafting antibodies and generating CDR variantsto optimize activity have been described previously (see, e.g.,international patent applications WO 98/33919; WO 00/78815; WO01/27160). The procedure can be performed to achieve grafting of donorCDRs and affinity reacquisition in a simultaneous process. The methodssimilarly can be used, either alone or in combination with CDR grafting,to modify or optimize the binding affinity of a variable region. Themethods for conferring donor CDR binding affinity onto an acceptorvariable region are applicable to both heavy and light chain variableregions and as such can be used to simultaneously graft and optimize thebinding affinity of an antibody variable region.

The donor CDRs can be altered to contain a plurality of different aminoacid residue changes at all or selected positions within the donor CDRs.For example, random or biased incorporation of the twenty naturallyoccurring amino acid residues, or preselected subsets, can be introducedinto the donor CDRs to produce a diverse population of CDR species.Inclusion of CDR variant species into the diverse population of variableregions allows for the generation of variant species that exhibitoptimized binding affinity for a predetermined antigen. A range ofpossible changes can be made in the donor CDR positions. Some or all ofthe possible changes that can be selected for change can be introducedinto the population of grafted donor CDRs. A single position in a CDRcan be selected to introduce changes or a variety of positions havingaltered amino acids can be combined and screened for activity.

One approach is to change all amino acid positions along a CDR byreplacement at each position with, for example, all twenty naturallyoccurring amino acids. The replacement of each position can occur in thecontext of other donor CDR amino acid positions so that a significantportion of the CDR maintains the authentic donor CDR sequence, andtherefore, the binding affinity of the donor CDR. For example, anacceptor variable region framework, either a native or alteredframework, can be grafted with a population of CDRs containing singleposition replacements at each position within the CDRs. Similarly, anacceptor variable region framework can be targeted for grafting with apopulation of CDRs containing more than one position changed toincorporate all twenty amino acid residues, or a subset of amino acids.One or more amino acid positions within a CDR, or within a group of CDRsto be grafted, can be altered and grafted into an acceptor variableregion framework to generate a population of grafted antibodies. It isunderstood that a CDR having one or more altered positions can becombined with one or more other CDRs having one or more alteredpositions, if desired.

A population of CDR variant species having one or more altered positionscan be combined with any or all of the CDRs which constitute the bindingpocket of a variable region. Therefore, an acceptor variable regionframework can be targeted for the simultaneous incorporation of donorCDR variant populations at one, two or all three recipient CDR locationsin a heavy or light chain The choice of which CDR or the number of CDRsto target with amino acid position changes will depend on, for example,if a full CDR grafting into an acceptor is desired or whether the methodis being performed for optimization of binding affinity.

Another approach for selecting donor CDR amino acids to change forconferring donor CDR binding affinity onto an antibody acceptor variableregion framework is to select known or readily identifiable CDRpositions that are highly variable. For example, the variable regionCDR3 is generally highly variable. This region therefore can beselectively targeted for amino acid position changes during graftingprocedures to ensure binding affinity reacquisition or augmentation,either alone or together with relevant acceptor variable frameworkchanges.

Murinized Antibodies:

An example of antibodies generated by grafting, as described above, aremurinized antibodies. As used herein, the term “murinized antibody” or“murinized immunoglobulin” refers to an antibody comprising one or moreCDRs from a human antibody of the present invention; and a humanframework region that contains amino acid substitutions and/or deletionsand/or insertions that are based on a mouse antibody sequence. The humanimmunoglobulin providing the CDRs is called the “parent” or “acceptor”and the mouse antibody providing the framework changes is called the“donor”. Constant regions need not be present, but if they are, they areusually substantially identical to mouse antibody constant regions, i.e.at least about 85-90%, preferably about 95% or more identical. Hence, insome embodiments, a full-length murinized human heavy or light chainimmunoglobulin contains a mouse constant region, human CDRs, and asubstantially human framework that has a number of “murinizing” aminoacid substitutions. Typically, a “murinized antibody” is an antibodycomprising a murinized variable light chain and/or a murinized variableheavy chain For example, a murinized antibody would not encompass atypical chimeric antibody, e.g., because the entire variable region of achimeric antibody is non-mouse. A modified antibody that has been“murinized” by the process of “murinization” binds to the same antigenas the parent antibody that provides the CDRs and is usually lessimmunogenic in mice, as compared to the parent antibody.

Antibody Fragments:

As used herein, the term “heavy chain portion” includes amino acidsequences derived from an immunoglobulin heavy chain. A polypeptidecomprising a heavy chain portion comprises at least one of: a CHIdomain, a hinge (e.g., upper, middle, and/or lower hinge region) domain,a CH2 domain, a CH3 domain, or a variant or fragment thereof. Forexample, a binding polypeptide for use in the invention may comprise apolypeptide chain comprising a CHI domain; a polypeptide chaincomprising a CHI domain, at least a portion of a hinge domain, and a CH2domain; a polypeptide chain comprising a CHI domain and a CH3 domain; apolypeptide chain comprising a CHI domain, at least a portion of a hingedomain, and a CH3 domain, or a polypeptide chain comprising a CHIdomain, at least a portion of a hinge domain, a CH2 domain, and a CH3domain. In another embodiment, a polypeptide of the invention comprisesa polypeptide chain comprising a CH3 domain. Further, a bindingpolypeptide for use in the invention may lack at least a portion of aCH2 domain (e.g., all or part of a CH2 domain). As set forth above, itwill be understood by one of ordinary skill in the art that thesedomains (e.g., the heavy chain portions) may be modified such that theyvary in amino acid sequence from the naturally occurring immunoglobulinmolecule.

In certain antibodies, or antigen-binding fragments, variants, orderivatives thereof disclosed herein, the heavy chain portions of onepolypeptide chain of a multimere are identical to those on a secondpolypeptide chain of the multimere. Alternatively, heavy chainportion-containing monomers of the invention are not identical. Forexample, each monomer may comprise a different target binding site,forming, for example, a bispecific antibody or diabody.

In another embodiment, the antibodies, or antigen-binding fragments,variants, or derivatives thereof disclosed herein are composed of asingle polypeptide chain such as scFvs and are to be expressedintracellularly (intrabodies) for potential in vivo therapeutic anddiagnostic applications.

The heavy chain portions of a binding polypeptide for use in thediagnostic and treatment methods disclosed herein may be derived fromdifferent immunoglobulin molecules. For example, a heavy chain portionof a polypeptide may comprise a CHI domain derived from an IgG1 moleculeand a hinge region derived from an IgG3 molecule. In another example, aheavy chain portion can comprise a hinge region derived, in part, froman IgG1 molecule and, in part, from an IgG3 molecule. In anotherexample, a heavy chain portion can comprise a chimeric hinge derived, inpart, from an IgG1 molecule and, in part, from an IgG4 molecule.

Thus, as also exemplified in the Examples, in one embodiment theconstant region of the antibody of the present invention or partthereof, in particular the CH2 and/or CH3 domain but optionally also theCHI domain is heterologous to the variable region of the native humanmonoclonal antibody isolated in accordance with the method of thepresent invention. In this context, the heterologous constant region(s)are preferably of human origin in case of therapeutic applications ofthe antibody of the present invention but could also be of for examplerodent origin in case of animal studies: see also the Examples.

As used herein, the term “light chain portion” includes amino acidsequences derived from an immunoglobulin light chain. Preferably, thelight chain portion comprises at least one of a VL or CL domain.

As previously indicated, the subunit structures and three dimensionalconfiguration of the constant regions of the various immunoglobulinclasses are well known. As used herein, the term “VH domain” includesthe amino terminal variable domain of an immunoglobulin heavy chain andthe term “CHI domain” includes the first (most amino terminal) constantregion domain of an immunoglobulin heavy chain. The CHI domain isadjacent to the VH domain and is amino terminal to the hinge region ofan immunoglobulin heavy chain molecule.

As used herein the term “CH2 domain” includes the portion of a heavychain molecule that extends, e.g., from about residue 244 to residue 360of an antibody using conventional numbering schemes (residues 244 to360, Kabat numbering system: and residues 231-340, EU numbering system:see Kabat EA et al. op. cit). The CH2 domain is unique in that it is notclosely paired with another domain. Rather, two N-linked branchedcarbohydrate chains are interposed between the two CH2 domains of anintact native IgG molecule. It is also well documented that the CH3domain extends from the CH2 domain to the C-terminal of the IgG moleculeand comprises approximately 108 residues.

As used herein, the term “hinge region” includes the portion of a heavychain molecule that joins the CHI domain to the CH2 domain. This hingeregion comprises approximately 25 residues and is flexible, thusallowing the two N-terminal antigen-binding regions to moveindependently. Hinge regions can be subdivided into three distinctdomains: upper, middle, and lower hinge domains; see Roux et al., JImmunol. 161 (1998), 4083.

As used herein the term “disulfide bond” includes the covalent bondformed between two sulfur atoms. The amino acid cysteine comprises athiol group that can form a disulfide bond or bridge with a second thiolgroup. In most naturally occurring IgG molecules, the CHI and CL regionsare linked by a disulfide bond and the two heavy chains are linked bytwo disulfide bonds at positions corresponding to 239 and 242 using theKabat numbering system (position 226 or 229, EU numbering system).

As used herein, the terms “linked”, “fused” or “fusion” are usedinterchangeably. These terms refer to the joining together of two moreelements or components, by whatever means including chemical conjugationor recombinant means. An “in-frame fusion” refers to the joining of twoor more polynucleotide open reading frames (ORFs) to form a continuouslonger ORF, in a manner that maintains the correct translational readingframe of the original ORFs. Thus, a recombinant fusion protein is asingle protein containing two or more segments that correspond topolypeptides encoded by the original ORFs (which segments are notnormally so joined in nature). Although the reading frame is thus madecontinuous throughout the fused segments, the segments may be physicallyor spatially separated by, for example, in-frame linker sequence. Forexample, polynucleotides encoding the CDRs of an immunoglobulin variableregion may be fused, in-frame, but be separated by a polynucleotideencoding at least one immunoglobulin framework region or additional CDRregions, as long as the “fused” CDRs are co-translated as part of acontinuous polypeptide. Accordingly, in one embodiment thepolynucleotide is a cDNA encoding the variable region and at least partof the constant domain. In one embodiment the polynucleotide is a cDNAencoding the variable region and the constant domain of an antibody ofthe present invention as defined herein.

Epitopes:

The minimum size of a peptide or polypeptide epitope for an antibody isthought to be about four to five amino acids. Peptide or polypeptideepitopes preferably contain at least seven, more preferably at leastnine and most preferably between at least about 15 to about 30 aminoacids. Since a CDR can recognize an antigenic peptide or polypeptide inits tertiary form, the amino acids comprising an epitope need not becontiguous, and in some cases, may not even be on the same peptidechain. In the present invention, a peptide or polypeptide epitoperecognized by antibodies of the present invention contains a sequence ofat least 4, at least 5, at least 6, at least 7, more preferably at least8, at least 9, at least 10, at least 15, at least 20, at least 25,between about 15 to about 30 or between about 30 to about 50 contiguousor non-contiguous amino acids of a molecule of interest of the presentinvention, i.e. at least one IFN-α subtype, or the homologous sequencesof the other IFN-α subtypes, in case the antibody recognizes more thanone subtype. For mapping of epitopes of IFN-α2 for the exemplaryantibodies 19D11 and 26B9 and of epitope of IFN-ω for the exemplaryantibody 26B9 of the present invention see FIG. 27.

Binding Characteristics:

By “binding” or “recognizing”, used interchangeably herein, it isgenerally meant that a binding molecule, e.g., an antibody binds to apredetermined epitope via its antigen-binding domain, and that thebinding entails some complementarity between the antigen-binding domainand the epitope. According to this definition, an antibody is said to“specifically bind” to an epitope when it binds to that epitope, via itsantigen-binding domain more readily than it would bind to a random,unrelated epitope. The term “specificity” is used herein to qualify therelative affinity by which a certain antibody binds to a certainepitope. For example, antibody “A” may be deemed to have a higherspecificity for a given epitope than antibody “B,” or antibody “A” maybe said to bind to epitope “C” with a higher specificity than it has forrelated epitope “D”. Unrelated epitopes are usually part of anonspecific antigen (e.g., BSA, casein, or any other specifiedpolypeptide), which may be used for the estimation of the bindingspecificity of a given binding molecule. In this respect, term “specificbinding” refers to antibody binding to a predetermined antigen with a Kothat is at least twofold less than its Ko for binding to a nonspecificantigen. The term “highly specific” binding as used herein means thatthe relative Ko of the antibody for the specific target epitope is atleast 10-fold less than the Ko for binding that antibody to otherligands.

Where present, the term “immunological binding characteristics,” orother binding characteristics of an antibody with an antigen, in all ofits grammatical forms, refers to the specificity, affinity,cross-reactivity, and other binding characteristics of an antibody.

By “preferentially binding”, it is meant that the binding molecule,e.g., antibody specifically binds to an epitope more readily than itwould bind to a related, similar, homologous, or analogous epitope.Thus, an antibody which “preferentially binds” to a given epitope wouldmore likely bind to that epitope than to a related epitope, even thoughsuch an antibody may cross-react with the related epitope. In respect ofparticular antigens, such as specific IFN-α subtypes the term“preferentially binding” means that the binding molecule, e.g., antibodyspecifically binds to an IFN-α subtype more readily than it would bindto a related, similar, homologous, or analogous IFN-α subtype.

By way of non-limiting example, a binding molecule, e.g., an antibodymay be considered to bind a first epitope preferentially if it bindssaid first epitope with dissociation constant (Ko) that is less than theantibody's Ko for the second epitope. In another non-limiting example,an antibody may be considered to bind a first antigen preferentially ifit binds the first epitope with an affinity that is at least one orderof magnitude less than the antibody's Ko for the second epitope. Inanother non-limiting example, an antibody may be considered to bind afirst epitope preferentially if it binds the first epitope with anaffinity that is at least two orders of magnitude less than theantibody's Ko for the second epitope.

In another non-limiting example, a binding molecule, e.g., an antibodymay be considered to bind a first epitope preferentially if it binds thefirst epitope with an off rate (k(off)) that is less than the antibody'sk(off) for the second epitope. In another non-limiting example, anantibody may be considered to bind a first epitope preferentially if itbinds the first epitope with an affinity that is at least one order ofmagnitude less than the antibody's k(off) for the second epitope. Inanother non-limiting example, an antibody may be considered to bind afirst epitope preferentially if it binds the first epitope with anaffinity that is at least two orders of magnitude less than theantibody's k(off) for the second epitope.

A binding molecule, e.g., an antibody or antigen-binding fragment,variant, or derivative disclosed herein may be said to bind a moleculeof interest of the present invention, a fragment or variant thereof withan off rate (k(off)) of less than or equal to 5×10−2 sec−1, 10−2 sec−1,5×10−3 sec−1 or 10−3 sec−1. More preferably, an antibody of theinvention may be said to bind a molecule of interest of the presentinvention or a fragment or variant thereof with an off rate (k(off))less than or equal to 5×10⁻⁴ sec⁻¹, 10⁻⁴ sec⁻¹, 5×10⁻⁵ sec⁻¹, or 10⁻⁵sec⁻¹ 5×10⁻⁶ sec⁻¹, 10⁻⁶ sec⁻¹, 5×10⁻⁷ sec−¹⁰ or 10⁻⁷ sec⁻¹.

A binding molecule, e.g., an antibody or antigen-binding fragment,variant, or derivative disclosed herein may be said to bind a moleculeof interest of the present invention or a fragment or variant thereofwith an on rate (k(on)) of greater than or equal to 10³ M⁻¹ sec⁻¹, 5×10³M⁻¹ sec⁻¹, 10⁴ M⁻¹ sec⁻¹ or 5×10⁴ M⁻¹ sec⁻¹. More preferably, anantibody of the invention may be said to bind a molecule of interest ofthe present invention or a fragment or variant thereof with an on rate(k(on)) greater than or equal to 10⁵ M⁻¹ sec⁻¹, 5×10⁵ M⁻¹ sec⁻¹, 10⁶ M⁻¹sec⁻¹, or 5×10⁶ M⁻¹ sec⁻¹ or 10⁷ M⁻¹ sec⁻¹. A binding molecule, e.g., anantibody is said to competitively inhibit binding of a referenceantibody to a given epitope if it preferentially binds to that epitopeto the extent that it blocks, to some degree, binding of the referenceantibody to the epitope. Competitive inhibition may be determined by anymethod known in the art, for example, competition ELISA assays. Anantibody may be said to competitively inhibit binding of the referenceantibody to a given epitope by at least 90%, at least 80%, at least 70%,at least 60%, or at least 50%.

As used herein, the term “affinity” refers to a measure of the strengthof the binding of an individual epitope with the CDR of a bindingmolecule, e.g., an immunoglobulin molecule; see, e.g., Harlow et al.,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,2nd ed. (1988) at pages 27-28. As used herein, the term “avidity” refersto the overall stability of the complex between a population ofimmunoglobulins and an antigen, that is, the functional combiningstrength of an immunoglobulin mixture with the antigen; see, e.g.,Harlow at pages 29-34. Avidity is related to both the affinity ofindividual immunoglobulin molecules in the population with specificepitopes, and also the valencies of the immunoglobulins and the antigen.For example, the interaction between a bivalent monoclonal antibody andan antigen with a highly repeating epitope structure, such as a polymer,would be one of high avidity. The affinity or avidity of an antibody foran antigen can be determined experimentally using any suitable method;see, for example, Berzofsky et al., “Antibody-Antigen Interactions” InFundamental Immunology, Paul, W. E., Ed., Raven Press New York, N.Y.(1984), Kuby, Janis Immunology, W H. Freeman and Company New York, N.Y.(1992), and methods described therein. General techniques for measuringthe affinity of an antibody for an antigen include ELISA, RIA, andsurface plasmon resonance. The measured affinity of a particularantibody-antigen interaction can vary if measured under differentconditions, e.g., salt concentration, pH. Thus, measurements of affinityand other antigen-binding parameters, e.g., Ko, ICso, are preferablymade with standardized solutions of antibody and antigen, and astandardized buffer.

Binding molecules, e.g., antibodies or antigen-binding fragments,variants or derivatives thereof of the invention may also be describedor specified in terms of their cross-reactivity. As used herein, theterm “cross-reactivity” refers to the ability of an antibody, specificfor one antigen, to react with a second antigen; a measure ofrelatedness between two different antigenic substances. Thus, anantibody is cross reactive if it binds to an epitope other than the onethat induced its formation. The cross reactive epitope generallycontains many of the same complementary structural features as theinducing epitope, and in some cases, may actually fit better than theoriginal.

For example, certain antibodies have some degree of cross-reactivity, inthat they bind related, but non-identical epitopes, e.g., epitopes withat least 95%, at least 90%, at least 85%, at least 80%, at least 75%, atleast 70%, at least 65%, at least 60%, at least 55%, and at least 50%identity (as calculated using methods known in the art and describedherein) to a reference epitope. An antibody may be said to have littleor no cross-reactivity if it does not bind epitopes with less than 95%,less than 90%, less than 85%, less than 80%, less than 75%, less than70%, less than 65%, less than 60%, less than 55%, and less than 50%identity (as calculated using methods known in the art and describedherein) to a reference epitope. An antibody may be deemed “highlyspecific” for a certain epitope, if it does not bind any other analog,ortholog, or homolog of that epitope.

Binding molecules, e.g., antibodies or antigen-binding fragments,variants or derivatives thereof of the invention may also be describedor specified in terms of their binding affinity to a molecule ofinterest of the present invention. Preferred binding affinities includethose with a dissociation constant or K_(D) less than 5×10⁻² M, 10⁻² M,5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M′ 10⁻⁵ M, 5×10⁻⁶ M, 10⁻⁶ M,5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M, 10⁻¹⁰M, 5×10⁻¹¹ M, 10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹M, 5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³M, 10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, or 10⁻¹⁵ M. Typically, theantibody binds with a dissociation constant (K_(D)) of 10⁻⁷ M or less toits predetermined antigen. Preferably, the antibody binds its cognateantigen with a dissociation constant (K_(D)) of 10−9 M or less and stillmore preferably with a dissociation constant (K_(D)) of 10⁻¹¹ M or less.

Modifications of Antibodies:

The immunoglobulin or its encoding cDNAs may be further modified. Thus,in a further embodiment the method of the present invention comprisesany one of the step(s) of producing a chimeric antibody, humanizedantibody, single-chain antibody, Fab-fragment, bi-specific antibody,fusion antibody, labeled antibody or an analog of any one of those.Corresponding methods are known to the person skilled in the art and aredescribed, e.g., in Harlow and Lane “Antibodies, A Laboratory Manual”,CSH Press, Cold Spring Harbor, 1988. When derivatives of said antibodiesare obtained by the phage display technique, surface plasmon resonanceas employed in the BIAcore system can be used to increase the efficiencyof phage antibodies which bind to the same epitope as that of any one ofthe antibodies provided by the present invention (Schier, HumanAntibodies Hybridomas 7 (1996), 97-io5; Malmborg, J Immunol. Methods 183(1995), 7-13). The production of chimeric antibodies is described, forexample, in international application WO89/09622. Methods for theproduction of humanized antibodies are described in, e.g., Europeanapplication EP-Al O 239 400 and international application WO90/07861.Further sources of antibodies to be utilized in accordance with thepresent invention are so-called xenogeneic antibodies. The generalprinciple for the production of xenogeneic antibodies such as humanantibodies in mice is described in, e.g., international applicationsWO91/10741, WO94/02602, WO96/34096 and WO 96/33735. As discussed above,the antibody of the invention may exist in a variety of forms besidescomplete antibodies; including, for example, Fv, Fab and F(ab)2, as wellas in single chains; see e.g. international application WO88/09344.

The antibodies of the present invention or their correspondingimmunoglobulin chain(s) can be further modified using conventionaltechniques known in the art, for example, by using amino aciddeletion(s), insertion(s), substitution(s), addition(s), and/orrecombination(s) and/or any other modification(s) known in the arteither alone or in combination. Methods for introducing suchmodifications in the DNA sequence underlying the amino acid sequence ofan immunoglobulin chain are well known to the person skilled in the art;see, e.g., Sambrook, Molecular Cloning A Laboratory Manual, Cold SpringHarbor Laboratory (1989) N.Y. and Ausubel, Current Protocols inMolecular Biology, Green Publishing Associates and Wiley Interscience,N.Y. (1994). Modifications of the antibody of the invention includechemical and/or enzymatic derivatizations at one or more constituentamino acids, including side chain modifications, backbone modifications,and N- and C-terminal modifications including acetylation,hydroxylation, methylation, amidation, and the attachment ofcarbohydrate or lipid moieties, cofactors, and the like. Likewise, thepresent invention encompasses the production of chimeric proteins whichcomprise the described antibody or some fragment thereof at the aminoterminus fused to heterologous molecule such as a label or a drug.Antigen binding molecules generated this way may be used for druglocalization to cells expressing the appropriate surface structures ofthe diseased cell and tissue, respectively. This targeting and bindingto cells could be useful for the delivery of therapeutically ordiagnostically active agents and gene therapy/gene delivery.Molecules/particles with an antibody of the invention would bindspecifically to cells/tissues expressing the particular antigen ofinterest, and therefore could have diagnostic and therapeutic use.

Samples:

As used herein, the term “sample” or “biological sample” refers to anybiological material obtained from a subject or patient. In one aspect, asample can comprise blood, cerebrospinal fluid (“CSF”), or urine. Inother aspects, a sample can comprise whole blood, plasma, mononuclearcells enriched from peripheral blood (PBMC) such as lymphocytes (i.e.T-cells, NK-cell or B-cells), monocytes, macrophages, dendritic cellsand basophils; and cultured cells (e.g., B-cells from a subject). Asample can also include a biopsy or tissue sample including tumortissue. In still other aspects, a sample can comprise whole cells and/ora lysate of the cells. In one embodiment a sample comprises peripheralblood mononuclear cells (PBMC). Samples can be collected by methodsknown in the art.

Identification of Anti-IFN-α Antibodies, Isolation of Corresponding BCells and Recombinant Expression of Anti-IFN-α Antibodies:

Identification of B-cells specific for the anti-IFN-α antibodies of thepresent invention, as enlisted in Table 1, and as exemplary shown forseveral IFN-α subtypes and molecular cloning of antibodies displayingspecificity of interest as well as their recombinant expression andfunctional characterization can be generally performed as described inthe international applications WO2013/098419 and WO2013/098420; seeExamples sections therein, in particular Examples 1 and 2 on pages 118to 120 of WO2013/098419 and Examples 1 to 4 on pages 27 to 31 ofWO2013/098420, the disclosure content of which is incorporated herein byreference.

Briefly, in one embodiment of the present invention cultures of singleor oligoclonal B-cells were cultured and the supernatant of the culture,which contains antibodies produced by said B-cells was screened forpresence and affinity of antibodies specific for one or more of theIFN-a subtypes, as described in the Examples. In another embodiment,patient sera were first screened for the presence of autoantibodiesagainst IFN-α subtypes and then those with high titer were selected forperipheral blood mononuclear cells isolation; see Example 2 on pages118-120 of WO2013/098419, the disclosure content of which isincorporated herein by reference. The screening process comprisesscreening for binding on fragments, peptides or derivatives of IFN-αsubtypes. Subsequently, the antibody for which binding is detected orthe cell producing said antibody were isolated; see Example 3 on page120 of WO2013/098419, the disclosure content of which is incorporatedherein by reference. Thus, a preliminary screen can be done on a panelof candidate donors, using samples containing antibody secreting cells(such as total peripheral blood or serum). In particular, mononuclearcells can be isolated from blood or lymphatic tissues using standardseparation techniques for isolating peripheral blood mononuclear cells(PBMCs), such as gradient centrifugation. After and/or before thisseparation step, the samples of sera (or plasma), cell culturesupernatants, or cells (obtained from different patients, from differenttissues, and/or at different time points) can be prescreened usingstandard technologies for detecting the presence of antibodies andantibody-secreting cells (e.g. ELISA, BIACORE, Western blot, FACS,SERPA, antigen arrays, neutralization of viral infection in a cellculture system, or ELISPOT assays). The literature provides severalexamples of these technologies showing, for example, the use of ELISPOTfor characterizing the immune response in vaccinated donors (Crotty etal., Immunol Meth. 286 (2004), 111-122), the use of antigen microarraysas diagnostic tools for newly infected patients (Mezzasoma et al., ClinChem. 48 (2002), 121-130, and other technologies for measuringantigen-specific immune responses (Kem et al., Trends Immunol. 26(2005), 477-484).

After identification of candidate anti-IFN-α antibodies and B cellssecreting them, respectively, the nucleic acid sequence that encodes theantibody of interest is obtained, comprising the steps of preparing a Bcell and obtaining/sequencing nucleic acid from the B cell that encodesthe antibody of interest and further inserting the nucleic acid into orusing the nucleic acid to prepare an expression host that can expressthe antibody of interest, culturing or sub-culturing the expression hostunder conditions where the antibody of interest is expressed and,optionally, purifying the antibody of interest. It goes without sayingthat the nucleic acid may be manipulated in between to introducerestriction sites, to change codon usage, and/or to add or optimizetranscription and/or translation regulatory sequences. These techniquesare state of the art and can be performed by the person skilled in theart without undue burden. For example, the heavy chain constant regioncan be exchanged for that of a different isotype or eliminatedaltogether. The variable regions can be linked to encode single chain Fvregions. Multiple Fv regions can be linked to confer binding ability tomore than one target or chimeric heavy and light chain combinations canbe employed. Once the genetic material is available, design of analogsas described above which retain both their ability to bind the desiredtarget is straightforward. Methods for the cloning of antibody variableregions and generation of recombinant antibodies are known to the personskilled in the art and are described, for example, in Gilliland et al.,Tissue Antigens 47 (1996), 1-20; Doenecke et al., Leukemia 11 (1997),1787-1792. In a preferred embodiment of the present invention however, Bcells are obtained and the corresponding antibody is expressed by themethods described in international application WO2013/098420, inparticular in Example 3, on pages 28-30 therein, the disclosure contentof which is incorporated herein by reference.

Diseases and Disorders:

Unless stated otherwise, the terms “disorder” and “disease” are usedinterchangeably herein. The term “autoimmune disorder” as used herein isa disease or disorder arising from and directed against an individual'sown tissues or organs or a co-segregate or manifestation thereof orresulting condition therefrom. Autoimmune diseases are primarily causedby dysregulation of adaptive immune responses and autoantibodies orautoreactive T cells against self-structures are formed. Nearly allautoimmune diseases have an inflammatory component, too.Autoinflammatory diseases are primarily inflammatory, and some classicautoinflammatory diseases are caused by genetic defects in innateinflammatory pathways. In autoinflammatory diseases, no autoreactive Tcells or autoantibodies are found. In many of these autoimmune andautoinflammatory disorders, a number of clinical and laboratory markersmay exist, including, but not limited to, hypergammaglobulinemia, highlevels of autoantibodies, antigen-antibody complex deposits in tissues,benefit from corticosteroid or immunosuppressive treatments, andlymphoid cell aggregates in affected tissues. Without being limited to atheory regarding B-cell mediated autoimmune disorder, it is believedthat B cells demonstrate a pathogenic effect in human autoimmunediseases through a multitude of mechanistic pathways, includingautoantibody production, immune complex formation, dendritic and T-cellactivation, cytokine synthesis, direct chemokine release, and providinga nidus for ectopic neo-lymphogenesis. Each of these pathways mayparticipate to different degrees in the pathology of autoimmunediseases.

As used herein, an “autoimmune disorder” can be an organ-specificdisease (i.e., the immune response is specifically directed against anorgan system such as the endocrine system, the hematopoietic system, theskin, the cardiopulmonary system, the gastrointestinal and liversystems, the renal system, the thyroid, the ears, the neuromuscularsystem, the central nervous system, etc.) or a systemic disease that canaffect multiple organ systems (for example, systemic lupus erythematosus(SLE), rheumatoid arthritis, polymyositis, autoimmune polyendocrinopathysyndrome type 1 (APS 1)/autoimmunepolyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) etc.Preferred such diseases include autoimmune rheumatologic disorders (suchas, for example, rheumatoid arthritis, Sjogren's syndrome, scleroderma,lupus such as SLE and lupus nephritis, polymyositis/dermatomyositis, andpsoriatic arthritis), autoimmune dermatologic disorders (such as, forexample, psoriasis, pemphigus group diseases, bullous pemphigoiddiseases, and cutaneous lupus erythematosus), and autoimmune endocrinedisorders (such as, for example, diabetic-related autoimmune diseasessuch as type 1 or insulin dependent diabetes mellitus (T1DM or IDDM),autoimmune thyroid disease (e.g., Graves' disease and thyroiditis)) anddiseases affecting the generation of autoimmunity such as autoimmunepolyendocrinopathy syndrome type 1 (APS1)/autoimmunepolyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) MyastheniaGravis (MG/Thymoma). Preferred diseases include, for example, SLE, RA,T1DM, MS, Sjogren's syndrome, Graves' disease, thyroiditis, andglomerulonephritis, and APS1. Still more preferred are RA, SLE, and MS,and mostly preferred SLE.

Labels and Diagnostics:

Labeling agents can be coupled either directly or indirectly to theantibodies or antigens of the invention. One example of indirectcoupling is by use of a spacer moiety. Furthermore, the antibodies ofthe present invention can comprise a further domain, said domain beinglinked by covalent or non-covalent bonds. The linkage can be based ongenetic fusion according to the methods known in the art and describedabove or can be performed by, e.g., chemical cross-linking as describedin, e.g., international application WO94/04686. The additional domainpresent in the fusion protein comprising the antibody of the inventionmay preferably be linked by a flexible linker, advantageously apolypeptide linker, wherein said polypeptide linker comprises plural,hydrophilic, peptide-bonded amino acids of a length sufficient to spanthe distance between the C-terminal end of said further domain and theN-terminal end of the antibody of the invention or vice versa. Thetherapeutically or diagnostically active agent can be coupled to theantibody of the invention or an antigen-binding fragment thereof byvarious means. This includes, for example, single-chain fusion proteinscomprising the variable regions of the antibody of the invention coupledby covalent methods, such as peptide linkages, to the therapeutically ordiagnostically active agent. Further examples include molecules whichcomprise at least an antigen-binding fragment coupled to additionalmolecules covalently or non-covalently include those in the followingnon-limiting illustrative list. Traunecker, Int. J Cancer Surp. SuDP 7(1992), 51-52, describe the bispecific reagent janusin in which the Fvregion directed to CD3 is coupled to soluble CD4 or to other ligandssuch as OVCA and IL-7. Similarly, the variable regions of the antibodyof the invention can be constructed into Fv molecules and coupled toalternative ligands such as those illustrated in the cited article.Higgins, J Infect. Disease 166 (1992), 198-202, described ahetero-conjugate antibody composed of OKT3 cross-linked to an antibodydirected to a specific sequence in the V3 region of GP120. Suchhetero-conjugate antibodies can also be constructed using at least thevariable regions contained in the antibody of the invention methods.Additional examples of specific antibodies include those described byFanger, Cancer Treat. Res. 68 (1993), 181-194 and by Fanger, Crit. Rev.Immunol. 12 (1992), 101-124. Conjugates that are immunotoxins includingconventional antibodies have been widely described in the art. Thetoxins may be coupled to the antibodies by conventional couplingtechniques or immunotoxins containing protein toxin portions can beproduced as fusion proteins. The antibodies of the present invention canbe used in a corresponding way to obtain such immunotoxins. Illustrativeof such immunotoxins are those described by Byers, Seminars Cell. Biol.2 (1991), 59-70 and by Fanger, Immunol. Today 12 (1991), 51-54.

The above described fusion protein may further comprise a cleavablelinker or cleavage site for proteinases. These spacer moieties, in turn,can be either insoluble or soluble (Diener et al., Science 231 (1986),148) and can be selected to enable drug release from the antigen at thetarget site. Examples of therapeutic agents which can be coupled to theantibodies and antigens of the present invention for immunotherapy arechemokines, homing molecules, drugs, radioisotopes, lectins, and toxins.The ¹²⁷I, ¹³¹I, ¹¹¹In, ¹⁷⁷Lu, ¹⁵O, ¹³N, ³²P, ³³P, ²⁰³Pb, ¹⁸⁶Re, ¹⁸⁸Re,¹⁰⁵Rh, ⁹⁷Ru, ³⁵S, ¹⁵³Sm and ^(99m)Tc. drugs with which can be conjugatedto the antibodies and antigens of the present invention depend on thedisease context in which the conjugated molecules are intended to beused. For example, antibodies specific for targets useful in treatmentof tumor diseases can be conjugated to compounds which are classicallyreferred to as anti-neoplastic drugs such as mitomycin C, daunorubicin,and vinblastine. In using radioisotopically conjugated antibodies orantigens of the invention for, e.g., tumor immunotherapy, certainisotopes may be more preferable than others depending on such factors asleukocyte distribution as well as stability and emission. Depending onthe autoimmune response, some emitters may be preferable to others. Ingeneral, a and B particle emitting radioisotopes are preferred inimmunotherapy. Preferred are short range, high energy a emitters such as²¹²Bi. Examples of radioisotopes which can be bound to the antibodies orantigens of the invention for therapeutic purposes are ¹²⁵I, ¹³¹I, ⁹⁰Y,⁶⁷Cu, ²¹²Bi, ²¹²At, ²¹¹Pb, ⁴⁷Sc, ¹⁰⁹Pd and ¹⁸⁸Re. Other therapeuticagents which can be coupled to the antibody or antigen of the invention,as well as ex vivo and in vivo therapeutic protocols, are known, or canbe easily ascertained, by those of ordinary skill in the art.Non-limiting examples of suitable radionuclides for labeling are ¹⁹⁸Au,²¹²Bi, ¹¹C, ¹⁴C, ⁵⁷Co, ⁶⁷Cu, ¹⁸F, ⁶⁷Ga, ³H, ¹⁶⁶Ho, ¹¹¹In, ^(113m)In,¹²³I, ¹²⁵I, ¹²⁷I, ¹³¹I, ^(III)In, ¹⁷⁷Lu, ¹⁵O, ¹³N, ³²P, ³³P, ²⁰³Pb,¹⁸⁶Re, ¹⁸⁸Re, ¹⁰⁵Rh, ⁹⁷Ru, ³⁵S, ¹⁵³Sm, and ^(99m)Tc. Other moleculessuitable for labeling are a fluorescent or luminescent dye, a magneticparticle, a metal, and a molecule which may be detected through asecondary enzymatic or binding step such as an enzyme or peptide tag.Commercial fluorescent probes suitable for use as labels in the presentinvention are listed in the Handbook of Fluorescent Probes and ResearchProducts, 8th Edition, the disclosure contents of which are incorporatedherein by reference. Magnetic particles suitable for use in magneticparticle-based assays (MPAs) may be selected from paramagnetic,diamagnetic, ferromagnetic, ferromagnetic and superpara-magneticmaterials.

General methods in molecular and cellular biochemistry useful fordiagnostic purposes can be found in such standard textbooks as MolecularCloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., HarborLaboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed.(Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollaget al., John Wiley & Sons 1996). Reagents, detection means and kits fordiagnostic purposes are available from commercial vendors such asPharmacia Diagnostics, Amersham, BioRad, Stratagene, Invitrogen, andSigma-Aldrich as well as from the sources given any one of thereferences cited herein, in particular patent literature.

Treatment and Drugs:

As used herein, the terms “treat” or “treatment” refer to boththerapeutic treatment and prophylactic or preventative measures, whereinthe object is to prevent or slow down (lessen) an undesiredphysiological change or disorder, such as the development of anautoimmune and/or autoinflammatory disease. Beneficial or desiredclinical results include, but are not limited to, alleviation ofsymptoms, diminishment of extent of disease, stabilized (i.e., notworsening) state of disease, delay or slowing of disease progression,amelioration or palliation of the disease state, and remission (whetherpartial or total), whether detectable or undetectable. “Treatment” canalso mean prolonging survival as compared to expected survival if notreceiving treatment. Those in need of treatment include those alreadywith the condition or disorder as well as those prone to have thecondition or disorder or those in which the manifestation of thecondition or disorder is to be prevented.

If not stated otherwise the term “drug,” “medicine,” or “medicament” areused interchangeably herein and shall include but are not limited to all(A) articles, medicines and preparations for internal or external use,and any substance or mixture of substances intended to be used fordiagnosis, cure, mitigation, treatment, or prevention of disease ofeither man or other animals; and (B) articles, medicines andpreparations (other than food) intended to affect the structure or anyfunction of the body of man or other animals; and (C) articles intendedfor use as a component of any article specified in clause (A) and (B).The term “drug,” “medicine,” or “medicament” shall include the completeformula of the preparation intended for use in either man or otheranimals containing one or more “agents,” “compounds”, “substances” or“(chemical) compositions” as in some other context also otherpharmaceutically inactive excipients as fillers, disintegrants,lubricants, glidants, binders or ensuring easy transport,disintegration, disaggregation, dissolution and biological availabilityof the “drug,” “medicine,” or “medicament” at an intended targetlocation within the body of man or other animals, e.g., at the skin, inthe stomach or the intestine. The terms “agent,” “compound” or“substance” are used interchangeably herein and shall include, in a moreparticular context, but are not limited to all pharmacologically activeagents, i.e. agents that induce a desired biological or pharmacologicaleffect or are investigated or tested for the capability of inducing sucha possible pharmacological effect by the methods of the presentinvention.

Examples of “anti-rheumatic drugs” and immunosuppressive drugs includechloroquine, hydroxycloroquine, myocrisin, auranofin, sulfasalazine,methotrexate, leflunomide, etanercept, infliximab (plus oral andsubcutaneous methotrexate), adalimumab etc., azathioprine,D-penicilamine, gold salts (oral), gold salts (intramuscular),minocycline, cyclosporine including cyclosporine A and topicalcyclosporine, tacrolimus, mycophenolate mofetil, cyclophosphamide,staphylococcal protein A (Goodyear and Silverman, J. Exp. Med., 197(2003), 125-39), including salts and derivatives thereof, etc.

Examples of “non-steroidal anti-inflammatory drugs” or “NSAIDs” includeaspmn, acetylsalicylic acid, ibuprofen and ibuprofen retard, fenoprofen,piroxicam, flurbiprofen, naproxen, ketoprofen, naproxen, tenoxicam,benorylate, diclofenac, naproxen, nabumetone, indomethacin, ketoprofen,mefenamic acid, diclofenac, fenbufen, azapropazone, acemetacin,tiaprofenic acid, indomethacin, sulindac, tolmetin, phenylbutazone,diclofenac and diclofenac retard, cyclooxygenase (COX)-2 inhibitors suchas GR 253035, MK966, celecoxib (CELEBREX®;4-(5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl),benzenesulfon-amide and valdecoxib (BEXTRA®), and meloxicam (MOBIC®),including salts and derivatives thereof, etc. Preferably, they areaspirin, naproxen, ibuprofen, indomethacin, or tolmetin. Such NSAIDs areoptionally used with an analgesic such as codenine, tramadol, and/ordihydrocodinine or narcotic such as morphine.

By “subject” or “individual” or “animal” or “patient” or “mammal,” ismeant any subject, particularly a mammalian subject, e.g., a humanpatient, for whom diagnosis, prognosis, prevention, or therapy isdesired.

Pharmaceutical Carriers:

Pharmaceutically acceptable earners and administration routes can betaken from corresponding literature known to the person skilled in theart. The pharmaceutical compositions of the present invention can beformulated according to methods well known in the art; see for exampleRemington: The Science and Practice of Pharmacy (2000) by the Universityof Sciences in Philadelphia, ISBN 0-683-306472, Vaccine Protocols. 2ndEdition by Robinson et al., Humana Press, Totowa, N.J., USA, 2003;Banga, Therapeutic Peptides and Proteins: Formulation, Processing, andDelivery Systems. 2nd Edition by Taylor and Francis. (2006), ISBN:0-8493-1630-8. Examples of suitable pharmaceutical carriers are wellknown in the art and include phosphate buffered saline solutions, water,emulsions, such as oil/water emulsions, various types of wetting agents,sterile solutions etc. Compositions comprising such carriers can beformulated by well-known conventional methods. These pharmaceuticalcompositions can be administered to the subject at a suitable dose.Administration of the suitable compositions may be effected by differentways. Examples include administering a composition containing apharmaceutically acceptable carrier via oral, intranasal, rectal,topical, intraperitoneal, intravenous, intramuscular, subcutaneous,subdermal, transdermal, intrathecal, and intracranial methods. Aerosolformulations such as nasal spray formulations include purified aqueousor other solutions of the active agent with preservative agents andisotonic agents. Such formulations are preferably adjusted to a pH andisotonic state compatible with the nasal mucous membranes.Pharmaceutical compositions for oral administration, such as singledomain antibody molecules (e.g., “nanobodies™”) etc are also envisagedin the present invention. Such oral formulations may be in tablet,capsule, powder, liquid or semi-solid form. A tablet may comprise asolid carrier, such as gelatin or an adjuvant. Formulations for rectalor vaginal administration may be presented as a suppository with asuitable carrier; see also O'Hagan et al., Nature Reviews, DrugDiscovery 2(9) (2003), 727-735. Further guidance regarding formulationsthat are suitable for various types of administration can be found inRemington's Pharmaceutical Sciences, Mace Publishing Company,Philadelphia, Pa., 17th ed. (1985) and corresponding updates. For abrief review of methods for drug delivery see Langer, Science 249(1990), 1527-1533.

Dosage Regimen:

The dosage regimen will be determined by the attending physician andclinical factors. As is well known in the medical arts, dosages for anyone patient depends upon many factors, including the patient's size,body surface area, age, the particular compound to be administered, sex,time and route of administration, general health, and other drugs beingadministered concurrently. A typical dose can be, for example, in therange of 0.001 to 1000 μg (or of nucleic acid for expression or forinhibition of expression in this range); however, doses below or abovethis exemplary range are envisioned, especially considering theaforementioned factors. Generally, the regimen as a regularadministration of the pharmaceutical composition should be in the rangeof 1 μg to 10 mg units per day. If the regimen is a continuous infusion,it should also be in the range of 1 μg to 10 mg units per kilogram ofbody weight per minute, respectively. Progress can be monitored byperiodic assessment. Preparations for parenteral administration includesterile aqueous or non-aqueous solutions, suspensions, and emulsions.Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, vegetable oils such as olive oil, and injectable organic esterssuch as ethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like. Furthermore, the pharmaceutical composition of theinvention may comprise further agents such as anti-tumor agents andcytotoxic drugs, depending on the intended use of the pharmaceuticalcomposition.

In addition, co-administration or sequential administration of otheragents may be desirable. A therapeutically effective dose or amountrefers to that amount of the active ingredient sufficient to amelioratethe symptoms or condition. Therapeutic efficacy and toxicity of suchcompounds can be determined by standard pharmaceutical procedures incell cultures or experimental animals, e.g., ED50 (the dosetherapeutically effective in 50% of the population) and LD50 (the doselethal to 50% of the population). The dose ratio between therapeutic andtoxic effects is the therapeutic index, and it can be expressed as theratio, LD50/ED50.

Preferably, the therapeutic agent in the composition is present in anamount sufficient for preventing inflammation or suppression of theimmune response.

These and other embodiments are disclosed and encompassed by thedescription and examples of the present invention. Further literatureconcerning any one of the materials, methods, uses and compounds to beemployed in accordance with the present invention may be retrieved frompublic libraries and databases, using for example electronic devices.For example the public database “Medline” may be utilized, which ishosted by the National Center for Biotechnology Information and/or theNational Library of Medicine at the National Institutes of Health.Further databases and web addresses, such as those of the EuropeanBioinformatics Institute (EBI), which is part of the European MolecularBiology Laboratory (EMBL) are known to the person skilled in the art andcan also be obtained using internet search engines. An overview ofpatent information in biotechnology and a survey of relevant sources ofpatent information useful for retrospective searching and for currentawareness is given in Berks, TIBTECH 12 (1994), 352-364.

The above disclosure generally describes the present invention. Severaldocuments are cited throughout the text of this specification. Fullbibliographic citations may be found at the end of the specificationimmediately preceding the claims. The contents of all citedreferences(including literature references, issued patents, publishedpatent applications as cited throughout this application andmanufacturer's specifications, instructions, etc.) are hereby expresslyincorporated by reference; however, there is no admission that anydocument cited is indeed prior art as to the present invention. A morecomplete understanding can be obtained by reference to the followingspecific examples which are provided herein for purposes of illustrationonly and are not intended to limit the scope of the invention.

EXAMPLES

The Examples 1 to 9 which follow and corresponding FIGS. 1 to 32 furtherillustrate the invention, but should not be construed to limit the scopeof the invention in any way. Detailed descriptions of conventionalmethods, such as those employed herein can be found in the citedliterature; see also “The Merck Manual of Diagnosis and Therapy”Seventeenth Ed. ed. by Beers and Berkow (Merck & Co., Inc., 2003).

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Methods inmolecular genetics and genetic engineering are described generally inthe current editions of Molecular Cloning: A Laboratory Manual,(Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, 2nded., Cold Spring Harbor Laboratory Press); DNA Cloning, Volumes I and II(Glover ed., 1985); Oligonucleotide Synthesis (Gait ed., 1984); NucleicAcid Hybridization (Hames and Higgins eds. 1984); Transcription AndTranslation (Hames and Higgins eds. 1984); Culture Of Animal Cells(Freshney and Alan, Liss, Inc., 1987); Gene Transfer Vectors forMammalian Cells (Miller and Calos, eds.); Current Protocols in MolecularBiology and Short Protocols in Molecular Biology, 3rd Edition (Ausubelet al., eds.); and Recombinant DNA Methodology (Wu, ed., AcademicPress). Gene Transfer Vectors For Mammalian Cells (Miller and Calos,eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols.154 and 155 (Wu et al., eds.); Immobilized Cells And Enzymes (IRL Press,1986); Perbal, A Practical Guide To Molecular Cloning (1984); thetreatise, Methods In Enzymology (Academic Press, Inc., N.Y.);Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker,eds., Academic Press, London, 1987); Handbook Of ExperimentalImmunology, Volumes I-IV (Weir and Blackwell, eds., 1986). Reagents,cloning vectors, and kits for genetic manipulation referred to in thisdisclosure are available from commercial vendors such as BioRad,Stratagene, Invitrogen, and Clontech. General techniques in cell cultureand media collection are outlined in Large Scale Mammalian Cell Culture(Hu et al., Curr. Opin. Biotechnol. 8 (1997), 148); Serum-free Media(Kitano, Biotechnology 17 (1991), 73); Large Scale Mammalian CellCulture (Curr. Opin. Biotechnol. 2 (1991), 375); and Suspension Cultureof Mammalian Cells (Birch et al., Bioprocess Technol. 19 (1990), 251.

Material and Methods

Patients selection, peripheral blood mononuclear cells (PBMC) isolationfrom APECED/APS1patients memory B cell culture and antibody isolationwere carried out as described in the international applicationsWO2013/098419 and WO2013/098420 with the difference that specificity ofthe antibodies isolated and analyzed was directed towards IFN-α subtypesas defined hereinabove and below instead of IL-17 and IL-22, which werespecifically used in the mentioned PCT applications; see Examplessections therein, in particular Examples 1 and 2 on pages 117 to 120 andExample 17 on pages 168-171 of WO2013/098419 and Examples 1 to 4 onpages 27 to 31 of WO2013/098420, the disclosure content of which isincorporated herein by reference.

The molecular cloning of human antibodies of the present invention andsubsequent antibody production and purification were performed asdescribed in the international application WO2013/098419, see theExamples section of the application and in particular Examples 1 to 3 onpages 117-120 therein, the disclosure content of which is incorporatedherein by reference. Mutation analysis of the AIRE gene was performed asdescribed in the international application WO2013/098419; see theExamples therein, in particular the “Mutation analysis of the AIRE gene”section in Materials and Methods of the Examples, on pages 115-116, thedisclosure content of which is incorporated herein by reference, withparticular steps performed as described in WO99/15559. In this concern,genotyping of the respective mutations in the AIRE (APECED) gene isperformed as described in international application WO99/15559 inExample 2 at pages 12 to 13; a confirmation of the mutations in exons 2and 6 of the AIRE gene as described in Example 3 of internationalapplication WO99/15559 at page 13, line 5 bridging to page 14, line 13,the disclosure content of which is incorporated herein by reference inits entirety. In particular, for the mutation analysis the DNA samplesare purified from peripheral blood mononuclear cells from patients withAPECED and from suspected carriers of APECED and from normal healthycontrols (according to Sambrook et al. 1989, Molecular Cloning. ALaboratory Manual. CSH Press) and subjected to PCR using primersspecific for all identified exons.

Example 1 Detection of Cytokine Specific Antibodies in the Serum ofPatients

The general presence of various cytokine and disease specific antibodiesin the sera of the patients suffering from the genetic condition APECED(Autoimmune polyendocrinopathy candidiasis epidermal dysplasia, alsocalled Autoimmune polyendocrinopathy type 1 (APS1)) has been determinedby protoarray analysis as described in Example 7 on page 128 andindicated Tables 1 and 2 on pages 128-130 of applicant's internationalapplication WO2013/098419, the disclosure content of which isincorporated herein by reference. Altogether sera from 23 patients,presented by codes from APS1-1 to APS1-23 were used in the assays. Eightcontrol sera were obtained from healthy laboratory personnel, agematched with the patients and coded as C1-C8. See FIG. 4 indicating thepresence of IFN-α subtype specific and dsDNA-specific antibodies in the23 patients examined. In addition to the seroreactivities shown in FIG.4, patients APS1-9 and APS1-2 have shown seroreactivity towardsIFN-α1/13, IFN-α5, IFN-α6, IFN-α10, IFN-α14, IFN-α16 and IFN-α21.

Anti-dsDNA antibodies are highly specific for SLE and used in thediagnosis of the disease. Surprisingly, no APS1 patients have lupusdespite the frequent presence of anti-dsDNA antibodies. However, APS1patients display pronounced seroreactivity against several interferon-asubtypes which are clinically-relevant drug targets involved in manylupus-implicated molecular mechanisms, implicating the possiblesuitability of these antibodies in SLE treatment.

ELISA-IFN-α

96 well microplates (Costar, USA) were coated with human IFN-α1, IFN-α2(ImmunoTools), IFN-α4 (SinoBiological), IFN-α5, IFN-α6, IFN-α8, IFN-α21(all form PBL) and IFN-α14 (ATGen) or IFN-α8 (Novus Biologicals). Plateswere washed with PBS-T and blocked 1 h at room temperature with PBScontaining 2% BSA (Sigma, Buchs, Switzerland). Patient sera, B cellconditioned medium, or recombinant antibody preparations were incubatedfor 2 h at room temperature. Binding of human IgG to the antigen ofinterest was determined using a horseradish peroxidase conjugated goatanti human Fe-gamma-specific antibody (Jackson ImmunoResearch, EuropeLtd., Cambridgeshire, UK) followed by measurement of the HRP activityusing a TMB substrate solution (TMB, Sigma, Buchs, Switzerland). Bindingto human Fe fusion mouse IFN-α2, IFN-α4, IFN-α14 (SinoBiological) wasdetected with horseradish peroxidase conjugated anti-F(ab′)2-specificantibody (see FIG. 19A).

Example 2 ECSO ELISA Determination of the Antibodies of the PresentInvention

EC50 binding of the hMABs of the present invention to IFN-α2(ImmunoTools), IFN-α4 (SinoBiological), IFN-α14 (ATGen) was determinedby ELISA (see also Table 3 below for details concerning the recombinantproteins used). Serial dilutions of MABs (from 1000 ng/ml down to 0.0169ng/ml) were incubated for 2 hours with antigen-coated plates (coatingovernight at 1 μg/ml in PBS, followed by wash out and blocking with 2%BSA in PBS). The plates were subsequently washed and binding of MABs wasdetected with anti-human HRP-conjugated secondary antibody.Concentrations of MAB resulting in half of maximal binding to respectiveantigens (EC50, ng/ml) were calculated using Prism 4 GraphPad softwareon sigmoidal dose-response curves (variable slope, 4 parameters)obtained by plotting the log of the concentration versus OD 450 nmmeasurements; see FIG. 3 and Table 4 below.

TABLE 3 List ofrecombinant proteins used in the Elisa assays. TargetProvider Catalog number IFN-alpha Immunotools 11343596 lbeta (IFN-αl/13)IFN-α2 ImmunoTools 11343516 IFN-α4 Sino Biological 10336-H08B IFN-α5 PBL11135 IFN-α6 PBL 11165 IFN-α8 PBL 11115 IFN-α21 PBL 11130 IFN-α14 ATGenATGP1500 IFN-omega 1 ProSpec CYT-040 IFN.gamma Immunotools 11343536gIFN-αl, 2, 4, In-house production, 5, 6, 7, 8, 10, Fusion constructwith 14, 16, 17, 21, B, W Gaussia luciferase

TABLE 4 Summary ofIC 50 neutralization values of exemplary anti-IFN-αantibodies 19D11, 26B9, 8Hl, 12H5 and 50El 1 of the present invention asobtained in the ISRE dual luciferase reporter assay. IC 50 (ng/ml)Antigen 19D11 26B9 8Hl 12HS SOEll IFN-αl 3.80 8.60 28.98 51.32 97.38IFN-α2 1.62 2.83 1039.0 10.66 158.6 IFN-α4 0.95 2.07 5.43 0.48 20.25IFN-α5 0.85 3.74 1.78 22.61 0.91 IFN-α6 0.79 3.16 77.70 3.55 39.30IFN-α7 0.37 1.57 0.90 0.64 1.90 IFN-α8 27.69 205.0 571.0 0.86 4.02IFN-αl0 0.72 1.84 2.69 0.66 3.63 IFN-α14 0.31 2.01 784.6 2.15 9.20IFN-α16 1.86 4013.0 1.86 32.99 2.35 IFN-α17 0.75 2.22 2.11 0.75 2.31IFN-α21 2.22 4.65 2.78 13.02 3.05 IFN-ω — 0.50 2.13 — 113.2

Previous EC50 binding versus IC50 neutralization assays performed inaccordance with the present invention of the exemplary anti-IFN-αantibodies using IFN-α-proteins obtained from commercial suppliers asindicated in Table 3 and IFN-α-proteins gIFN-α2/-4 and -14 generated inaccordance with the present invention gave similar results.

In addition, binding of exemplary MABs 19D11, 25C3, 26B9, 5D1 and 13B11of the present invention to IFN-α1 (ImmunoTools) was compared to thebinding of these antibodies to IFN-α2 (ImmunoTools) by ELISA. In thisexperiment, examined MABs (300 ng/ml) were incubated for 2 hours withantigen-coated plates (coating overnight at 1 μg/ml in PBS, followed bywash out and blocking with 2% BSA in PBS). The plates were subsequentlywashed and binding of MABs was detected with anti-human HRP-conjugatedsecondary antibody. Exemplary MABs 19D11, 25C3, 26B9 and 13B11 haveshown a comparable binding affinity to IFN-α1 and IFN-α2 (see FIG. 11A).However, exemplary anti-IFN-α MAB 5D1 binds IFN-α2 and does notcross-react with IFN-α1 (see FIG. 11A). In a further experiment bindingof exemplary MABs 19D11, 25C3, 26B9, 5D1 and 13B11 of the presentinvention to IFN-α8 was compared by LIPS to the binding of theseantibodies to IFN-α14 (both IFN-Gaussia luciferase fusion proteins).Herein, exemplary MAB 13B11 did not show cross-reactivity with IFN-α8.The other exemplary antibodies19D11, 25C3, 26B9 and 5D1 have shownstronger binding of INFA8 than of IFN-α14 (see FIG. 11B). In anadditional experiment binding of exemplary MABs 5D1, 13B11 19D11, 25C3,26B9 and 31B4 of the present invention to IFN-α5, IFN-α6, IFN-α8,IFN-α21 (all from PBL) and IFN-α14 (ATGen) was determined andcomparised. Exemplary anti-IFN-α-a antibody 13B1 did not cross-reactwith IFN-α8 and IFN-α21 and antibody 19D11 cross-reacted with a loweraffinity with IFN-α21 than with the other IFN-α subtypes (see FIG. 11C).

Example: 3

Neutralization Assays

The neuralizing assays are carried out on cell lines that respond to thestudied cytokine, i.e. carry the necessary receptor. The ligand bindingto receptor activates a corresponding signaling pathway, translocationof transcription factors to the nucleus and upregulate responder genetranscription, translation and if applicable product secretion. Thecytokine concentration used is selected from the beginning of the linearpart of the dose-response curve to maximize the sensitivity of theassay. To test the neutralizing capacity of antibodies the optimalconcentration of the target cytokine is preincubated with serialdilutions of serum, supernatant or purified antibody samples. Theresults are expressed as titer or concentration of antibody that showthe value half-way between the positive and negative controls.

Phospho-STATJ Assay

30,000 HEK 293T or HEK 293T MSR cells were seeded intoPoly-L-Lysine-coated 96-well plates (BD Biocoat, Bedford, Mass., USA) orinto regular tissue culture-treated 96-well plates (Cat. No. 3598,Corning Inc., Corning, N.Y., USA), respectively. The following day,rhIFN-as or supernatants of HEK 293T cells transiently expressingIFN-Gaussia luciferase fusion proteins (g1 IFNs) were mixed withanti-IFN-α mAbs or control IgG (5 μg/ml) and preincubated for one hourat 37° C. After preincubation, the mixtures were used to stimulate HEK293T or HEK 293T MSR cells for 10 min at 37° C. Following stimulation,cells were lysed with CelLytic™ M lysis buffer supplemented withprotease and phosphatase inhibitors (Cat. No. C2978, P5726, P0044,P8340, SIGMA-ALDRICH, St. Louis, Mo., USA) and the collected lysateswere cleared at 13,000 RPM, 4° C. In a tabletop centrifuge. Lysates weresubjected to reducing SDS-PAGE and blotted onto nitrocellulosemembranes. Membranes were blocked with a buffer containing 0.25% bovinegelatin, 150 mM NaCl, 5 mM EDTA, 50 mM Tris/HCl pH 7.5, 0.05% TritonX-100 for one hour at room temperature, followed by incubation withrabbit monoclonal antibodies against phosphorylated STAT1 (Tyr701,diluted 1:1000 in blocking buffer, Cat. No. 9167, Cell SignalingTechnology, Danvers, Mass., USA) at 4° C. over night. On the next day,blots were washed three times with blocking buffer followed byincubation with horseradish peroxidase-linked secondary antibodiesagainst rabbit IgG (diluted 1:20,000 in blocking buffer, Cat. No.111-035-144, Jackson ImmunoResearch, West Grove, Pa., USA). After threeadditional washing steps, an ECL substrate was added (Cat. No. 34087,Thermo Fisher Scientific, Rockford, Ill., USA) and reactive bands werevisualized via autoradiography. Bound antibodies were removed byincubation in Restore Western Blot Stripping Buffer (Cat. No. 21059,Thermo Fisher Scientific) and a rabbit polyclonal anti-STAT1 serum wasused to visualize total STAT1 levels (diluted 1:1000 in blocking buffer,Cat. No. 9172, Cell Signaling Technology).

In the following the results of two Phospho-STAT1 assays performed withexemplary anti-IFN-a specific antibodies 5D1, 19D11, 25C3, 26B9, 31B4and 13B11 are discussed. As may be seen in FIGS. 5B and 5C, addition ofexemplary anti-IFN-α specific antibodies 19D11, 25C3, 26B9, 31B4 and13B11 prohibits the IFN-α2, IFN-α4 and IFN-α14 dependent phosphorylationof STAT1 indicating the IFN-α neutralizing capacity of the antibodies ofthe present invention. In addition, exemplary anti-IFN-α specificantibodies 19D11, 26B9 and 31B4 also prohibit the IFN-α1, g1 IFN-α5 andIFN-α6 dependent phosphorylation of STAT1, with the neutralizationcapacity of antibody 26B9 slightly reduced against IFN-α5. Antibody 25C3showed a slightly weaker neutralization capacity concerning IFN-α6 andIFN-α1 4 activity in comparison to its neutralizing capacity towardsIFN-α2 and IFN-α4. Addition of exemplary anti-IFN-α specific antibody5D1 of the present invention showed neutralizing activity of thisantibody against IFN-α2 and IFN-α4, wherein no or very weakneutralization could be observed in respect of IFN-α14 dependentSTAT1-phosphorylation (FIG. SC, right panel) and a slightly weakerneutralization of IFN-α6.

After IFN-α1b or IFN-α16 stimulation exemplary antibodies 19D11, 25C3,26B9, 31B4 and 13B11 have shown neutralization capacity towards IFN-α1b(IFN-α1/13) activity, wherein no neutralization could be observed inrespect of antibody 5D1 (FIG. 5B). Concerning IFN-α16, only antibodies19D11, 5D1 and 13B11 have shown neutralization capacity, whereinexemplary antibodies 25C3, 26B9 and 31B4 indicated at least severelyreduced or even a lack of neutralizing capacity towards IFN-α16 (FIG.5C). Exemplary antibody 13B11 potently neutralized IFN-α1, IFN-α2,IFN-α4 and g1 IFN-α5, whereas very weak neutralization could be observedin respect of IFN-α6 dependent STAT1 phosphorylation. rhIFN-αconcentration as used was: 10 ng/ml (IFN-α1b), 2 ng/ml (IFN-α2, IFN-α4,IFN-α6, IFN-α16). g1 IFN-α5 supernatant was used at its EC 80 dilution.

While all exemplary antibodies except 13B11 neutralized IFN-α21 in thisfunctional assay (FIG. 5D), exemplary antibodies 25C3 and 13B11 did notneutralize IFN-α6 (FIG. 5B). MABs concentration was at 5 μg/ml, rhIFN-αs(PBL) at 2 ng/ml. All antibodies were neutralizing IFN-α5 (g1IFN-α5)(FIG. 5B). Antibodies 25C3 and 13B11 did not neutralize IFN-α8(g1IFN-α8). None of the exemplary anti-IFN-α antibodies of the presentinvention antibodies neutralized IFN-y (IFN-gamma/IFNG) (FIG. 5D). Otherthan in the above general experimental description, here HEK 293T cellswere either left untreated (−) or stimulated with supernatants of HEK293T cells transiently expressing human IFN-Gaussia luciferase fusionproteins (+) in the absence of antibodies or in the presence of 5 μg/mlhuman-derived exemplary human MABs or a human control IgG (huigG) asindicated in FIG. 5D.

In a second experimental round, additional IFN-α subtypes have beentested. The results of these two experimental rounds have been combinedin FIG. 5. After IFN-α7, g1 IFN-α8, IFN-α10, IFN-α14 or IFN-α16stimulation exemplary antibody 19D11 has shown neutralization capacitytowards all tested IFNs, wherein exemplary antibodies 26B9 and 31B4could not neutralize IFN-α16 (FIG. 5C). Exemplary antibody 25C3 fullyneutralized IFN-α7, but showed weaker neutralization of g1 IFN-α8,IFN-α10, IFN-α14 and IFN-α16. Exemplary antibody 5D1 neutralized IFN-α7,g1 IFN-α8, IFN-α10 and IFN-α16 but no neutralization of IFN-α14.Exemplary antibody 13B11 showed neutralization of IFN-α7, IFN-α10,IFN-α14 and IFN-α16, but no neutralization of g1 IFN-α8. rhIFN-αconcentration as used was 2 ng/ml (IFN-α7, IFN-α10, IFN-α14, IFN-α16).g1 IFN-α8 supernatant was used at its EC 80 dilution.

All exemplary antibodies neutralized IFN-α17 in this functional assay(FIG. 5D). All exemplary antibodies except 13B11 neutralized IFN-α21.Only two exemplary antibodies, 26B9 and 31B4, neutralized IFN-ω in thisfunctional assay, while none of the exemplary antibodies neutralizedIFNB or g1 IFNG. rhIFN-α concentration was 2 ng/ml, g1 IFNG supernatantwas used at its EC 80 dilution.

ISRE-Luciferase Reporter Assay

20,000 HEK 293T or 10,000 HEK 293T MSR cells were seeded inPoly-L-Lysine-coated 96-well plates (BD Biocoat) (in white half-area96-well plates (Cat. No. 3688, Corning Inc.)—values in brackets indicatethe differences in the second experimental option with HEK 293 TMSRcells) and reverse-transfected with 100 ng (50 ng) of premixedISRE-Firefly luciferase reporter and Renilla luciferase constructs (Cat.No. CCS-008L, Qiagen, Hilden, Germany) (see scheme of the constructs inFIG. 6A) using Fugene HD according to the manufacturer's instructions(Promega, Madison, Wis., USA). The Renilla luciferase-expressingconstruct served as an internal normalization control. Cells wereincubated overnight in Opti-MEM® I Reduced Serum Medium supplementedwith 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 10% (0.5%)fetal bovine serum (Life Technologies, Carlsbad, Calif., USA) at 37° C.,5% CO2 in a humidified atmosphere. Following overnight incubation, cellswere stimulated for 24 hours with medium containing mixtures of rhIFN-aswith or without anti-IFN-α mAbs or control IgG that had beenpreincubated for one hour at 37° C. After 24 hours of stimulation, dualluciferase reporter assays were performed according to themanufacturer's instructions (Promega).

As may be seen in FIG. 7, addition of exemplary anti-IFN-α specificantibodies 19D11, 25C3, 26B9, 31B4 (FIGS. 7A and B) and 13B11 (FIGS. 7Cand D) prohibits the IFN-α2, IFN-α4 and IFN-α14 dependentISRE-Luciferase reporter activation indicating the IFN-α neutralizingcapacity of the antibodies of the present invention. Addition ofexemplary anti-IFN-α specific antibody 5D1 of the present inventionshowed neutralizing activity of this antibody against IFN-α2 and IFN-α4,wherein no or very weak neutralization could be observed in respect ofIFN-α14 dependent ISRE-Luciferase activation (FIGS. 7C and D). Asalready seen in the phospho-STATI-assay, antibody 25C3 showed a slightlyweaker neutralization capacity concerning IFN-α14 activity in comparisonto its neutralizing capacity towards IFN-α2 and IFN-α4. Similarly,exemplary anti-IFN-α specific antibody 13B11 has shown a slightly weakerneutralization capacity towards IFN-α14 (FIGS. 7C and D). Exemplaryantibody 8H1 neutralizes IFN-ω, together with IFN-α1, A4, A5, A6, A7,A10, A16, A17 and A21, while showing weaker neutralization of IFN-α2, A8and A14 (FIG. 7E). Exemplary antibody 12H5 strongly inhibitsISRE-Luciferase reporter induction by all alpha interferons, namelyIFN-α1, A2, A4, A5, A6, A7, A8, A10, A14, A16, A17 and A21, whilevirtually not affecting IFN-ω-mediated reporter induction. Neitherexemplary antibody 8H1 nor 12H5 interfere with IFNB-mediated reporterinduction (FIG. 7E).

As shown in FIG. 8, exemplary antibody 26B9 potently neutralizesrhIFN-α1, A2, A4, A5, A6, A8, A10, A14, A17, A21 and rhIFN-ω, whileweakly neutralizing rhIFN-α16. rhIFN concentration was 10 ng/ml(IFN-α1), 1.3 ng/ml (IFN-α16) and 2 ng/ml (all other rhIFNs). A summaryof the IC 50 values as determined in the ISRE-Luciferase reporter assayis shown in Table 4.

As may be seen in FIG. 9, exemplary antibody 19D11 neutralizes allrhIFN-α molecules, namely IFN-αI, A2, A4, A5, A6, A7, A8, A10, A14, A16,A17 and A21. rhIFN concentration was 10 ng/ml (IFN-α1), 1.3 ng/ml(IFN-α16) and 2 ng/ml (all other rhIFNs). A summary of the IC 50 valuesas determined in the ISRE-Luciferase reporter assay is shown in Table 4.

Chemiluminescent Cellular Binding Assay

30,000 HEK 293T MSR cells were seeded in white half-area 96-well tissueculture plates (Cat. No. 3688, Corning Inc.). The following day,supernatants of HEK 293T cells transiently expressing human IFN-Gaussialuciferase fusion proteins were mixed with anti-IFN mAbs, control IgG orexcess concentrations of unlabeled recombinant IFN-α2 and preincubatedfor one hour at 37° C. After preincubation, the mixtures were used tostimulate HEK 293T MSR cells for 40 minutes at 37° C. Upon binding,cells were washed three times with PBS, and the Gaussia luciferase assaywas developed using the Gaussia Flash Assay Kit according to themanufacturer's instructions (Cat. No. 16159, Thermo Fisher Scientific).

As shown in FIG. 10C, exemplary antibody 19D11 efficiently neutralizesbinding of g1 IFN-α2, A4, A5, A6, A7, A8, A10, A14, A16, A17 and A21 toHEK 293T MSR cells endogenously expressing IFN receptors. In contrast,exemplary antibody 19D11 does virtually not interfere with binding of g1IFNB and g1 IFN-ω. Binding of all g1 IFNs showed herein is apparentlynot affected by a control human antibody (huigG). Antibodyconcentration: 5 μg/ml.

As may be seen in FIG. 10D, binding of g1 IFN-α16 to HEK 293T MSR cellsis more efficiently neutralized by exemplary antibody 19D11 as comparedto exemplary antibody 26B9. However, exemplary antibody 26B9 stronglyneutralizes binding of g1 IFN-ω to HEK 293T MSR cells, whereas exemplaryantibody 19D11 shows no apparent neutralization capacity towards thisligand. Binding of both g1 IFN-α16 and g1 IFN-ω remains unaffected by acontrol human antibody (huigG). Antibody concentration: 10 μg/ml.

Example 4 Validation of Subject Antibodies

Antibodies provided by the present invention are tested in concern oftheir neutralizing activity towards human IFN-α in animal diseasemodels. When performing such experiments it has to be ensured that humanIFN-α subtypes induce diseased phenotypes in mice and that nocross-reaction occurs between the tested IFN-α antibodies of the presentinvention and the murine IFN-α homologues. Since no adequate modelsystem for IFN-alpha was available in the prior art, the present Exampledescribes and provides such a system to test IFN-alpha neutralizingantibodies that do not cross react with mouse IFNs. First, the effect ofdifferent IFN-α subtypes was tested in vivo for the induction of earinflammation. In particular the proinflammatory activity of humansubtypes IFN-α2a, IFN-α2b, IFN-α4 and IFN-α14 have been tested. IFN-α2adiffers from IFN-α2b by amino acid 23 (Lys in IFN-α2a and Arg inIFN-α2b)

Ear Inflammation Assay

Ear inflammation phenotype was induced in 8 weeks old C57BL/6J (WT; fromCharles River) mice by intradermal injection of human IFN-α2a, IFN-α2b,IFN-α4 and IFN-α14 in 20 μl of PBS, or PBS control into each ear givenon alternate days at Day 0, Day 2 and Day 4 (20 μl/ear, 500 ng/ear, 1 μgtotal/mouse/day) using a 30-gauge needle. The mice were sacrificed atday 6; see Table 5 below and FIG. 14A for the experimental timeline.

TABLE 5 Group allocation of animals to the different cytokines tested.Group n Cytokine ng/20 μ1 A 5 PBS Na B 5 IFN-α2a 500 C 5 IFN-α2b 500 D 5IFN-α4 500 E 5 IFN-α14 500 n - number of animals in the group, ng/20 μ1-amount of cytokine injected per ear.

To test the proinflammatory effect of the injected IFN-α subtypes earthickness measurements of the animals were taken with a Mitutoyo digitalmicrometer during the cytokine administration by 2 measurements per earprior to cytokine injection at Day 0 and at alternate days at Day 1, Day3, Day 5 (indicated by letter Min FIG. 14A) and alternatively or inaddition at Day 6 after sacrifice of the animal

Furthermore, body weight is monitored during the treatment, to observeany possible weight changes due to the inflammation induction or itsrespective reduction due to the treatment applied. In addition, aftersacrifice of the animals H&E ((hematoxylin and eosin; see Harris, H. F.,J. Appl. Microscopy III(1900), 777-781 and Mallory, F. B.: Pathologicaltechnique. Philadelphia, Saunders, 1938.) histology stainings of theears are performed.

All four human IFN-α subtypes tested were able to significantly induceear swelling following cytokine injection; see FIGS. 14-16 and resultsof the experiment summarized in the table in FIG. 17. All ears weremarkedly thicker than PBS treated ears, this was significant in allgroups after the 2nd intradermal injection, from Day 3 until the end ofthe experiment. IFN-α14 was the most potent especially at Day 5 in thisexperiment. IFN-α2a and IFN-α4 induced similar levels of ear thickening.IFN-α2b was most similar to both IFN-α4 and IFN-α14 induced swelling.IFN-α2b induced swelling more than the IFN-α2a isoform in thisexperiment; see FIGS. 14-16 and the experimental results summary in thetable in FIG. 17.

The results of this experiment show the applicability of the earinflammation assay for tests of the therapeutic applicability of theantibodies of the present invention. Since the exemplary anti-IFN-αantibodies 19D11, 26B9, 31B4, 5D1 and 13B11 of the present invention didnot show any apparent cross-reaction with at least murine IFN-α subtypes2, 4 and 14 (see FIG. 19A), they are tested in the above indicated assayin respect of their neutralization properties towards human IFN-α usedfor induction of inflammation. Apparent binding affinity of exemplaryanti-IFN-α antibody 25C3 towards murine INFA2 and the affinity ofexemplary anti-IFN-α antibodies 5D1 and 19D11 towards murine IFN-α1 istaken into consideration when designing the in vivo CytoEarneutralization experiments described herein.

Such treatment tests are performed with the exemplary anti-IFN-αantibodies of the present invention by injection of the antibodies atdifferent time points during the above drafted experimental timeline(see also FIG. 14A) for testing induction of inflammation by humanIFN-α. For example, for testing the preventive and/or therapeutic effectone or more of the exemplary antibodies of the present invention areinjected together with or separately to the IFN-α subtype or subtypes atDay 0 of the experiment. In addition or alternatively, one or more ofthe antibodies of the present invention or IFN-α binding fragmentsthereof are injected on alternate days with the IFN-α subtype orsubtypes. For example, if the IFN-α subtype or subtypes are injected asindicated above at Days 0, 2 and 4 (short arrows in FIG. 14A), theantibodies are injected on the alternate Days 1, 3 and/or 5 (long arrowsin FIG. 14A).

The neutralizing potential of the antibodies of the present invention orIFN-α binding fragments thereof to reduce the induced ear inflammationphenotype and/or to prevent such an induction is examined by comparisonof ear swelling (thickness) observed in animals obtaining the anti-IFN-αantibody treatment and the control groups obtaining either PBS or humanIgGs of a binding specificity directed towards other molecules thanhuman IFN-α subtypes (of IFN-α non-related binding specificity).

Furthermore, or alternatively body weight is monitored during thetreatment, to observe any possible weight changes due to theinflammation induction or its respective reduction due to the treatmentapplied. In addition, after sacrifice of the animals H&E (hematoxylinand eosin; see supra) histology stainings of the ears are performed.This assay is used preferably as a surrogate model for psoriasis.

In a second experimental round, the above indicated assay has been usedwith some modifications to test neutralization properties of exemplaryantibodies 26B9 and 19D11 of the present invention towards inflammationinduced in mice ears by injections of human IFN-α14 (FIG. 28), IFN-α5(FIG. 29) and IFN-ω (FIG. 30). As may be seen from the time schemesshown in FIGS. 28A, 29A and 30A the experimental time line has beenprolonged here to 10 days with tested antibodies and controls injectedat experimental day 0 (IP), intradermal cytokine injections at days 1,3, 6 and 8 and a sacrifice of the test animals at day 10. Groupallocations of animals to the different cytokines tested and therespective concentrations and amounts of the cytokines, respectiveantibodies tested are indicated in tables in panels B of the respectivefigures. Both antibodies, 26B9 and 19D11 have shown pronouncedpreventive and/or therapeutic potential due to a significant reductionof the ear thickness on several experimental days after IFN-α14 (FIGS.28D and E) and IFN-α5 (FIGS. 29D and E) induced ear inflammation incomparison to IgG controls. Treatment with the reference IFN-α specificantibody (Ref. A in FIGS. 28, 29, in particular FIGS. 28F and 29F) ledalso to a significant reduction of ear swelling after IFN-α14 treatmenton several experimental days, however with a slightly lower reduction atday 10 compared with antibodies 26B9 and 19D11 of the present invention(compare curves F for 26B9 in FIGS. 28D and G for 19D11 in FIG. 28E withcurve H for Ref. A in FIG. 28F and with curve E in each FIG. for theIgG-control). Furthermore, exemplary antibody 26B9 has shown asignificant reduction of IFN-ω induced ear swelling at experimental day9 (FIG. 30D), wherein injections of antibody 19D11 and Ref A did notshow any significant reduction of the ear swelling compared tonon-specific IgG treatment. Accordingly, antibodies provided with thepresent invention have a high potential for use in prevention and/ortreatment of diseases associated with enhanced IFN-α and/or IFN-ωactivity.

CytoAnkle Assay:

In this assay mice cohorts (c57/b16, 7-8 weeks) are intraarticular (IA)injected with 62.5-io00 ng cytokine, e.g., at least one IFN-α subtypesuch as IFN-α2a, IFN-α2b, IFN-α4 or IFN-α14 or mixtures of several IFN-αsubtypes in 10 ul of PBS (or PBS control) into ankles every 48-72 hours.Axial ankle thickness measurements are than taken with a Mitutoyodigital micrometer. Animals are weighed each day and respective IFN-αsubtype or subtypes are administered while the mice are anaesthetizedwith isofluorane. The experimental time frame is designed as indicatedabove for the ear inflammation assay, with injections of the anti-IFN-αantibody or antibodies of the present invention, respective the controlgroups obtaining either PBS or human IgGs of IFN-α non-related bindingspecificity as indicated above. Reduction of the ankle swelling is usedas readout of the therapeutic effect of the antibodies of the presentinvention. This assay is used preferably as a surrogate model forarthritis, e.g., rheumatoid arthritis.

Example 5 Epitope Mapping of Exemplary IFN-α Antibodies

As a first step of mapping, differential binding of anti-IFN-α MABs ofthe present invention to distinct antigen binding sites was examined todetermine the number of different binding sites. For this purpose, MABswere expressed either with human (hMAB) or mouse (hmMAB) Fe andcross-competition experiments were carried out by coating antigen onplates and by detecting binding of hmMABs in the presence of largeexcess of human MABs. Detection of hmMABs bound to the ligand wasperformed by a HRP-conjugated secondary antibody directed against the Feportion of the primary antibody.

As may be seen from FIG. 2A and Table 6A below, exemplary anti-IFN-αantibodies 19D11, 26B9, 31B4 and 13B11 of the present invention competeeach other for binding of IFN-α2 but not with antibodies 5D1 and 25C3,indicating that 5D1 and 25C3 bind other site(s) of IFN-α2 than 19D11,26B9, 31B4 and 13B11. The same competition pattern may be seen forIFN-α4 and IFN-α14 with the difference however that 13B1 does notcompete for binding of IFN-α4 and only weakly competes for binding ofIFN-α14 with 19D11, 26B9, 31B4 indicating that the epitope theantibodies are binding to may be not conserved in this IFN-α subtypesand in particular divergent in IFN-α4. The results of the first approachalso indicate a weak competition of each other of anti-IFN-α antibodies5D1 and 25C3 of the present invention for binding of IFN-α2 and IFN-α4indicating a possible partially overlapping epitope of these antibodies;see FIGS. 2A, 2B and Tables 5A, 5B. Furthermore, a diverging competitionpattern on IFN-α14 may be observed, where hMAB 25C3 shows strongcompetition with hmMAB 5D1 but only a weak competition in the reversesituation (hMAB 5D1 against hmMAB 25C3) may be observed, indicating apossible preference of the 25C3 antibody towards an IFN-α14-specificepitope; see FIG. 2C and Table 6C below.

TABLE 6 Results of the cross-competition experiments of exemplaryantibodies of the present invention. Human MAB (hMAB) were added inlarge excess to plates coated with the respective antigens beforeaddition of MABs with mouse Fe (hmMAB). Human MAB competitor 19D11 25C326B9 31B4 5D1 13B11 A Human INF-α2 Competition 19D11 +++++ − +++++ +++++− ++++ of binding 25C3 − +++++ − − + − of hmMAB 31B4 +++++ − +++++ +++++(+) ++++ 5D1 − (+) − − +++(+) − 13B11 − − − − − +++(+) B Human INF-α4Competition 19D11 +++++ − ++++ +++(+) − − of binding 25C3 − ++++ − − (+)− of hmMAB 31B4 +++++ − +++++ +++++ − +++ 5D1 − (+) − − +++(+) − 13B11 −− − − − +(+) C Human INF-α14 Competition 19D11 +++++ − ++++ +++(+) − ++of binding 25C3 (+) +++++ (+) − +++ (+) of hmMAB 31B4 +++++ − ++++++++++ ++ ++(+) 5D1 +++++ +++++ +++++ +++++ +++++ (+) 13B11 +++(+) −+++(+) +++ − ++++

Binding regions of MABs to their respective antigens can be mapped by,e.g., PepStar™ analysis. Therefore, overlapping 20mer peptides (15 aminoacid overlap) are designed to cover the IFN-α-a subtypes of interest,e.g., IFN-α2, IFN-α4 and IFN-α14 including all known variants. Thepeptides and full length antigen (as positive control) are spotted onmicroarray and the peptide microarray is incubated with the primaryantibody followed by a fluorescently labelled secondary antibodydirected against the Fe portion of the primary antibody. To avoid falsenegatives caused by steric hindrance, an optimized hydrophilic linkermoiety is inserted between the glass surface and the antigen derivedpeptide sequence.

Such peptide mapping has been performed for exemplary antibodies 19D11and 26B9 of the present invention on peptide arrays of 18mer peptides ofhuman IFN-α2, respective human IFN-αW. The results of the assay areshown in FIG. 27. Exemplary antibody 19D11 binds specifically topeptides 19 (SAAWDETLLDKFYTELYQ SEQ ID NO: 99) and 32 (RITLYLKEKKYSPCAWEV SEQ ID NO: 100) of IFN-α2 (FIG. 27B). The antibody 26B9 bindsspecifically to peptide 22 (YTELYQQLNDLEACVIQG SEQ ID NO: 101) of IFN-α2(FIG. 27C) and to peptide 23 (TG1HQQLQHLETCLLQVV SEQ ID NO: 102) ofIFN-αW (FIG. 27D).

Example 6 Determination of MABs Binding to IFN-α Subtypes by LIPS Assay

In addition to the ELISA assay, binding of MABs to the different IFN-αsubtypes was determined by the LIPS assay. IFN-α5-, IFN-α6- andIFN-α8-Gaussia fusion proteins were produced by cloning IFN-α5, IFN-α6and IFN-α8 each fused with Gaussia luciferase at N-terminus andexpressing them individually by transient transfection of HEK293 cells(harvest of the supernatant after 2 days) as described in Example 10 onpage 158 and Example 15 on pages 165-167 in applicant's internationalapplication WO2013/098419, the disclosure content of which isincorporated herein by reference, using primers indicated in Table 8below. MABs (4 μg/ml) were diluted in Buffer A (50 mM Tris, pH 7.5, 100mM NaCl, 5 mM MgCl2, 1% Triton X-100) and incubated for 1 hour with anequal Protein A agarose beads (Exalpha) in wells of MultiScreen HTSFilter Plates (Millipore) on rotary shaker. Two volumes of IFN-α5- orIFN-α6- or IFN-α8-Gaussia fusion protein (1 million LU) were added andplates incubated for 1 hour. Plates were washed 5 times with Buffer Aand 2 additional times with PBS before addition of the substrate(Gaussia Luciferase Flash Assay Kit, Pierce) Luminescence (CPS) was readusing EnSprire (Perkin Elmer) (see FIGS. 11B, 12A-C and 13). A humanantibody binding to an unrelated antigen human MAB has been used asnegative control.

As can be seen from the results shown in FIGS. 11 and 12, exemplary MAB13B11 binds to: IFN-α2, 4, 5, 6, 10 and 14 but not, or weaker toIFN-α1/13 and not to IFN-α8. Furthermore, the results provided inexperiments described herein, e.g., in Examples 2 and 3 show thatexemplary MAB 13B11 neutralizes: IFN-α2, 4, 5, 14 but not IFN-α6, 8 andIFN-α21. All results concerning the binding and neutralizationproperties of the exemplary antibodies of the present invention asprovided by the experiments described in Examples 2, 3 and 6 aresummarized in Table 7 below.

TABLE 7 Binding (B) and neutralization (N) of different IFN subtypes byexemplary antibodies of the present invention as obtained in ELISA, LIPSor neutralization assays described herein. 19D11 25C3 26B9 31B4 5D113B11 Antigen B N B N B N B N B N B N IFN-α1 +++ +++ +++ +++ +++ +++ ++++++ − − +++ +++ IFN-α2 +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++IFN-α4 +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ IFN-α5 +++ ++++++ +++ +++ +++ +++ +++ +++ +++ +++ +++ IFN-α6 +++ +++ +++ + +++ +++ ++++++ +++ ++ +++ + IFN-α7 nd +++ nd +++ nd +++ nd +++ nd +++ nd +++ IFN-α8+++ +++ +++ + +++ +++ +++ +++ +++ +++ − − IFN-α10 +++ +++ +++ + +++ ++++++ +++ +++ +++ +++ +++ IFN-α14 +++ +++ +++ ++ +++ +++ +++ +++ +++ − ++++++ IFN-α16 +++ +++ +++ + +++ − +++ − +++ +++ +++ +++ IFN-α17 nd +++ nd+++ nd +++ nd +++ nd +++ nd +++ IFN-α21 +++ +++ +++ +++ +++ +++ +++ ++++++ +++ +++ − IFN-ω − − nd − +++ +++ nd +++ nd − nd − Exemplary humananti-IFN-α antibody of the present invention 19D11 neutralizes all IFN-αsubtypes and not IFN-ω. Exemplary antibody 26B9 neutralizes IFN-ω andall IFN-α subtypes except IFN-αI 6. +/++/+++ = binding, respectiveneutralisation; − = lack of binding/neutralization. nd = not determined;B = binding, determined by ELISA with commercial recombinant proteins orby LIPS with self-produced IFN-Gaussia luciferase fusion proteins; N =neutralization, determined by ISRE-Luciferase reporter assay or byphospho-STATI assay.

Previous experiments on binding and neutralization of different IFN-αsubtypes by the exemplary antibodies of the present invention in ELISA,LIPS and neutralization assays gave similar results.

TABLE 8 Overview of all IFN proteins and used primer sequences used forcloning the Gaussia-luciferase-IFN-fusion constructs. Primer Gene AAName Sequence and SEQ ID NO: IFN-α1 24- IFN-α1FTTTGGATCCTATGTGATCTCCCTGAGACCCACAGCCTGGA SEQ ID NO: 45 189 IFN-α1RTTTGCGGCCGCGACCAGATGTTATTCCTTCCTCCTTAATCTTTC SEQ ID NO: 46 IFN-α2 24-IFN-α2F TTTGGGATCCTCTGTGATCTGCCTCAAACCCACA SEQ ID NO: 47 188 IFN-α2RTTTGCGGCCGCTTACTTCTTAAACTTTCTTGCA SEQ ID NO: 48 IFN-α4 24- IFN-α4FTTTGGATCCTATGTGATCTGCCTCAGACCCACAGCCTGG SEQ ID NO: 49 189 IFN-α4RTTTGCGGCCGCTCAATCCTTCCTCCTTAATCTTTTTTGCAAGTTTGT SEQ ID NO: 50 TGAAAACIFN-α5 22- IFN-α5F TTTGGATCCTACTGGGCTGTGATCTGCCTCAGACCCACAGCCTGAGSEQ ID NO: 51 189 IFN-α5RTTTGCGGCCGCTCATTCCTTCCTCCTTAATCTTTCTTGCAAGTTTGC SEQ ID NO: 5 IFN-α6 21-IFN-α6F TTTGGATCCTATCTCTGGACTGTGATCTGCCTCAGACCCACAGCCTG SEQ ID NO: 5 189GGTC IFN-α6R TTTGCGGCCGCTTATTCCTTCCTCCTTAACCTTTCTTGCAAGTTTCSEQ ID NO: 54 IFN-α7 24- IFN-α7F TTTGGATCCTATGTGATCTGCCTCAGACCCACAGCCTGCSEQ ID NO: 55 189 IFN-α7RTTTGCGGCCGCGAACCAGTTTTCAATCCTTCCTCCTTAATCCTTTTTT SEQ ID NO: 56 IFN-α823- IFN-α8F TTTGGGATCCTCTGTGATCTGCCTCAGACTCACA SEQ ID NO: 57 189 IFN-α8RTTTGCGGCCGCTCATTCCTTACTCTTCAATCTT SEQ ID NO: 58 IFN-α10 24- IFN-α9FTTTGGATCCTATGTGATCTGCCTCAGACCCACAGCCTGGG SEQ ID NO: 59 89 IFN-α9RTTTGCGGCCGCTCAATCCTTCCTCCTTAATCTTTTTTGCAAGTTTGT SEQ ID NO: 50 TGAAAACIFN-α14 24- IFN-α14F TTTGGATCCTATGTAATCTGTCTCAAACCCACAGCCTGAASEQ ID NO: 60 189 IFN-α14RTTTGCGGCCGCTCAATCCTTCCTCCTTAATCTTTTTTGCAAGTTTGT SEQ ID NO: 61 IFN-α1624- IFN-α16F TTTGGATCCTATGTGATCTGCCTCAGACT SEQ ID NO: 62 189 IFN-α16RTTTGCGGCCGCTCAATCCTTCCTTCTTAATCC SEQ ID NO: 63 IFN-α17 24- IFN-α17FTTTGGATCCTATGTGATCTGCCTCAGACCCACAGCCTGGG SEQ ID NO: 59 189 IFN-α17RTTTGCGGCCGCGTTGAACCAGTTTTCAATCCTTCCTCCTTAATA SEQ ID NO: 64 IFN-α21 24-IFN-α21F TTTGGATCCTATGTGATCTGCCTCAGACCCACAGCCT SEQ ID NO: 65 189IFN-α21R TTTGCGGCCGCTCATTCCTTCCTCCTTAATCTTTCTTGAAAAA SEQ ID NO: 6 IFNB22- IFNB1F TTTGGATCCTAATGAGCTACAACTTGCTTGGATTCCTAC SEQ ID NO: 67 187IFNB1R TTTGCGGCCGCTCAGTTTCGGAGGTAACCTGTAAGTCT SEQ ID NO: 68 IFNG 24-IFNGF TTTGGATCCTACAGGACCCATATGTAAAAGAAGCAGAAAAC SEQ ID NO: 69 166 IFNGRTTTGCGGCCGCCCATTACTGGGATGCTCTTCGACCT SEQ ID NO: 70

Cloning Human IFN Subtypes in Fusion to Gaussia Luciferase

Coding sequences of IFN-α1, IFN-α6, IFN-α7, IFN-α8, IFN-α10, IFN-α14,IFN-α16, IFN-α17, IFN-α21, IFN-ω, IFNB, IFNG, IFNE and IFNK withoutsignal peptides were cloned into modified pPK-CMV-F4 fusion vector(PromoCell GmbH, Heidelberg; Germany) downstream of naturally secretedGaussia luciferase (Glue) that was replaced to the plasmid instead ofFirefly luciferase.

Example: 7 IC 50 Analysis of Exemplary Human-Derived IFN-α mAbs by ISRELuciferase Reporter Neutralization Assay

HEK 293T MSR cells (Cat. No. R79507, Invitrogen, Carlsbad, Calif., USA)transiently expressing ISRE-Firefly luciferase reporter and Renillaluciferase constructs (Cat. No. CCS-008L, Quiagen, Hilden, Germany) werestimulated with 2 ng/ml rhIFN-α2, rhIFN-α4, rhIFN-α14 or withsupernatants of HEK 293T cells transiently expressing human IFN-Gaussialuciferase fusion proteins (IFN-α5, IFN-α8), in the presence ofhuman-derived IFN-α mAbs 26B9 (FIG. 8A-E), 25C3 (FIG. 9A-E) or 19D11(FIG. 10A-E) as indicated. After 24 hours of stimulation, dualluciferase reporter assays were performed according to themanufacturer's instructions (Promega, Madison, Wis., USA).

In a confirmatory experimental round HEK 293T MSR cells transientlyexpressing ISRE-Firefly luciferase reporter and Renilla luciferaseconstructs as described above were stimulated with 10 ng/ml rhIFN-α1, 2ng/ml rhIFN-α2, rhIFN-α4, rhIFN-α5, rhIFN-α6, rhIFN-α8, rhIFN-α10,rhIFN-α14, rhIFN-α17, rhIFN-α21, 1.3 ng/ml rhIFN-α16, in the presence ofhuman-derived IFN-α mAbs 26B9 (FIG. 8F-R) or 19D11 (FIG. 10F-Q) asindicated. After 24 hours of stimulation, dual luciferase reporterassays were performed according to the manufacturer's instructions(Promega, Madison, Wis., USA). The same experimental setup has beenfurther used to perform the IC 50 analysis of human-derived mAbs 8H1(FIG. 20), 12H5 (FIG. 21) and 50E11 (FIG. 22). The results of the assaysare summarized in Table 4 above.

Example: 8 Antibody Affinity Measurements Using Surface PlasmonResonance (SPR) Technology

For affinity determination of the antibodies of the present inventionSPR measurements are performed using a ProteOn™ XPR36 instrument,according to the instructions of the manufacturer (Bio-RAD; HerculesCalif., USA) using the molecules of interest of the present invention inan analogous experimental setup as described in Example 14 ofinternational application WO2013/098419 on pages 163-165, the disclosurecontent of which is incorporated herein by reference. Alternatively orin addition a similar analysis is made using Biacore SPR instrumentsaccording to the manufacturer's instructions.

Results for the SPR measurements performed on exemplary antibodies 19D11and 26B9 of the present invention are shown in FIG. 26, with a 1:1binding kinetic observed for the antibodies towards IFN-α2b, IFN-α4,IFN-α14 and in respect of antibody 26B9 also towards IFN-ω. Theaffinities towards human IFN-α4 and IFN-α14 are in the sub-picomolarrange and in sub-nanomolar range for IFN-α2b. 26B9 also binds humanIFN-ω with a sub-picomolar affinity.

Example 9 Chemiluminescent Cellular Binding Assays

Interferons-Gaussia Luciferase

30,000 HEK 293T MSR cells were seeded in white half area 96-well tissueculture plates (Cat. No. 3688, Corning Inc.). The following day,supernatants of HEK 293T cells transiently expressing human IFN-Gaussialuciferase fusion proteins were mixed with anti-IFN mAbs, control IgG orexcess concentrations of unlabeled recombinant IFN-α2 and preincubatedfor one hour at 37° C. After preincubation, the mixtures were used tostimulate HEK 293T MSR cells for 40 minutes at 37° C. Upon binding,cells were washed three times with PBS, and the Gaussia luciferase assaywas developed using the Gaussia Flash Assay Kit according to themanufacturer's instructions (Cat. No. 16159, Thermo Fisher Scientific).

Binding to Transmembrane Antibodies

30,000 HEK 293T MSR cells were seeded in white half area 96-well tissueculture plates (Cat. No. 3688, Corning Inc.). During seeding, cells weretransfected with 100 ng cDNA encoding a transmembrane version ofanti-IFN mAb 26B9 (26B9-TM) using Fugene HD (Cat. No. E2311, Promega,Madison, Wis., USA). Surface antibody (26B9-TM) expression was analyzed48 hours after transfection in a cell-based ELISA (FIG. 24A).Forty-eight hours following transfection, supernatants of HEK 293T cellstransiently expressing human IFN-ω-Gaussia luciferase fusion proteins(g1 IFN-ω) were used to stimulate the previously transfected HEK 293TMSR cells for 40 minutes at 37° C. Alternatively, the g1 IFN-ωsupernatants were mixed with anti-IFN mAbs or control IgG andpreincubated for one hour at 37° C. After preincubation, the mixtureswere used to stimulate HEK 293T MSR cells transiently expressing 26B9-TMfor 40 minutes at 37° C. Upon binding, cells were washed three timeswith PBS, and the Gaussia luciferase assay was developed using theGaussia Flash Assay Kit according to the manufacturer's instructions(Cat. No. 16159, Thermo Fisher Scientific) showing that g1 IFN-ω wasspecifically binding to cells expressing 26B9-TM (FIG. 24B).

Crosscompetition Assay of Anti-IFN-ω Antibodies.

Above experimental setup has been also used to test crosscompetitionbetween exemplary antibodies 26B9, 31B4 and 8H1 of the present invention(see FIG. 25). HEK 293T MSR cells were reverse-transfected with cDNAencoding 26B9-TM. Forty-eight hours after transfection, g1 IFN-ω wasmixed and preincubated for one hour with soluble anti-IFN-ω antibodies26B9, 31B4, 8H1 or a control IgG (huIgG). Following incubation, themixtures were added to the transfected cells and binding was analysed inthe chemiluminescent cellular binding assay. Binding of g1 IFN-ω to26B9-TM is competed dose-dependently by soluble 26B9 and by the clonallyrelated 31B4 antibody. In contrast, binding is not affected by a controlIgG or by exemplary anti-IFN-ω antibody 8H1. These results indicate thatexemplary antibodies 26B9 and 3 1B4 share similar epitopes, while 8H1appears to bind to a distinct epitope.

The invention claimed is:
 1. A recombinant human monoclonalanti-interferon-alpha (IFN-α) antibody or an IFN-α binding fragmentthereof, wherein the antibody or IFN-α-binding fragment thereofcomprises: a variable heavy (VH) chain sequence comprising thesequence of (SEQ ID NO: 18)EVQLLESGAEVKRPGSSVRVSCRASGDTFSSYPISWVRQAPGQGLEWMGRILPALGVTNYAQNFRGRITITADKSPLTAYLELSSLRFEDTAVYYCASPSADIIPSILGTTLFAFWGQGSLVTVSS anda variable light (VL) chain sequence comprising the sequence of(SEQ ID NO: 20) EIVLTQSPGTLSLSPGEGATLSCRASQNVSRHYLTWYQQKPGQSPRLLIYGGSSRAIGVPDRFSGGGSGTDFTLTISRLEPEDFAVFYCQSYHSPPPVYT FGQGTKVEIK,

wherein the antibody has the following properties: (i) binds to humanIFN-α subtypes IFNα1/13(IFNαlb), IFNα2, IFNα4, IFNα5, IFNα6, IFNα7,IFNα8, IFNα10, IFNα14, IFNα16, IFNα17 and IFNα21; and (ii) does not bindto IFN-ω.
 2. The recombinant human monoclonal anti-interferon-alpha(IFN-α) antibody or an IFN-α binding fragment thereof of claim 1,wherein amino acids 1-6 and 121-126 of (SEQ ID NO: 18) have not beenisolated or sequenced from a human.
 3. The recombinant human monoclonalanti-interferon-alpha (IFN-α) antibody or an IFN-α binding fragmentthereof of claim 2, wherein amino acids 1-6 and 121-126 of (SEQ ID NO:18) are obtained from a database.
 4. The recombinant human monoclonalanti-interferon-alpha (IFN-α) antibody or an IFN-α binding fragmentthereof of claim 1, wherein amino acids 1-6 and 105-110 of (SEQ ID NO:20) have not been isolated or sequenced from a human.
 5. The recombinanthuman monoclonal anti-interferon-alpha (IFN-α) antibody or an IFN-αbinding fragment thereof of claim 4, wherein amino acids 1-6 and 105-110(SEQ ID NO: 20) are obtained from a database.
 6. The recombinant humanmonoclonal anti-interferon-alpha (IFN-α) antibody or an IFN-α bindingfragment thereof of claim 1, which is an IgG1 isotype.
 7. Therecombinant human monoclonal IFN-α antibody or IFN-α binding fragmentthereof of claim 1, further comprising a C_(H) and/or C_(L) constantregion comprising an amino acid sequence selected from SEQ ID NOs.:72and
 74. 8. The recombinant human monoclonal IFN-α antibody or IFN-αbinding fragment thereof of claim 1, which has an IC₅₀ value of <10 ngfor at least 10 human IFN-α subtypes.
 9. A composition comprising therecombinant human monoclonal IFN-α antibody or IFN-α binding fragmentthereof of claim
 1. 10. The composition of claim 9, further comprising apharmaceutically acceptable carrier.
 11. A method of producing therecombinant human monoclonal IFN-α antibody of claim 1 comprising thesteps of: (a) culturing in a cell culture a host cell comprising avector comprising a cDNA molecule or a polynucleotide encodinga variable heavy (VH) chain sequence comprising the sequence of(SEQ ID NO: 18) EVQLLESGAEVKRPGSSVRVSCRASGDTFSSYPISWVRQAPGQGLEWMGRILPALGVTNYAQNFRGRITITADKSPLTAYLELSSLRFEDTAVYYCASPSADIIPSILGTTLFAFWGQGSLVTVSS anda variable light (VL) chain sequence comprising the sequence of(SEQ ID NO: 20) EIVLTQSPGTLSLSPGEGATLSCRASQNVSRHYLTWYQQKPGQSPRLLIYGGSSRATGVPDRFSGGGSGTDFTLTISRLEPEDFAVFYCQSYHSPPPVYT FGQGTKVEIK,

under conditions allowing the expression of the antibody encoded by thecDNA molecule or the polynucleotide; and (b) isolating the antibody fromthe cell culture.
 12. A method of treating an inflammatory or autoimmunedisease or condition associated with the expression of IFN-α comprisingadministering to a subject a therapeutically effective amount of therecombinant human monoclonal anti-IFN-α antibody or IFN-α bindingfragment thereof of claim
 1. 13. The method of claim 12, wherein themethod comprises administering to the subject a therapeuticallyeffective dose of the recombinant human monoclonal IFN-α antibody orIFN-α binding fragment thereof.
 14. The method of claim 12, wherein therecombinant human monoclonal IFN-α antibody or IFN-α binding fragmentthereof is administered intravenously.
 15. The method of claim 14,wherein the recombinant human monoclonal IFN-α antibody or IFN-α bindingfragment thereof is administered at a dose of between 1 μg to 10 mg perday.
 16. The method of claim 15, wherein the recombinant humanmonoclonal IFN-α antibody or IFN-α binding fragment thereof isadministered by an infusion.
 17. The method of claim 16, wherein therecombinant human monoclonal IFN-α antibody or IFN-α binding fragmentthereof is administered by infusion at a dose of between 1 1tg to 10 mgunits per kilogram of body weight per minute.
 18. A method of treatingan inflammatory or autoimmune disease or condition associated with theexpression of IFN-α comprising administering to a subject atherapeutically effective amount of a composition comprising therecombinant human monoclonal anti-IFN-α antibody or IFN-α-bindingfragment thereof of claim
 1. 19. The method of claim 18, wherein themethod comprises administering to the subject a therapeuticallyeffective dose of the composition.
 20. The method of claim 18, whereinthe composition is administered intravenously.
 21. The method of claim20, wherein composition is administered at a dose of between 1 μg to 10mg units per day.
 22. The method of claim 21, wherein the composition isadministered by infusion.
 23. The method of claim 22, wherein thecomposition is administered by infusion at a dose of between 1 μg to 10mg units per kilogram of body weight per minute.
 24. A recombinant humanmonoclonal IFN-α antibody or IFN-α binding fragment thereof producedaccording to a method comprising the steps of: (a) culturing a pluralityof B cells obtained from a subject having autoimmune polyendocrinopathysyndrome type 1 (ASP1) or autoimmune polyendocrinopathy-candidiasisectodermal dystrophy (APECED) under conditions allowing only a definitelife span of the B cells; (b) identifying a B cell reactive to humanIFN-α subtypes IFNα1/13 (IFNαlb), IFNα2, IFNα4, IFNα5, IFNα6, IFNα7,IFNα8, IFNαl 0, IFNα14, IFNα16, IFNα17 and IFNα21 and not reactive toIFN-ω; (c) sequencing an antibody produced from the B cell; and (d)cloning the antibody of (c) to produce a recombinant antibody using oneor more primers, wherein the one or more primers introduce a sequencevariation into the recombinant antibody compared to the sequence of (c),wherein the IFN-α antibody or IFN-α-binding fragment thereof comprises:a variable heavy (VH) chain sequence comprising the sequence of(SEQ ID NO: 18) EVQLLESGAEVKRPGSSVRVSCRASGDTFSSYPISWVRQAPGQGLEWMGRILPALGVTNYAQNFRGRITITADKSPLTAYLELSSLRFEDTAVYYCASPSADIIPSILGTTLFAFWGQGSLVTVSS anda variable light (VL) chain sequence comprising the sequence of(SEQ ID NO: 20) EIVLTQSPGTLSLSPGEGATLSCRASQNVSRHYLTWYQQKPGQSPRLLIYGGSSRATGVPDRFSGGGSGTDFTLTISRLEPEDFAVFYCQSYHSPPPVY TFGQGTKVEIK.


25. The recombinant human monoclonal IFN-α antibody or IFN-α bindingfragment thereof of claim 24, wherein the conditions allowing only adefinite life span of the B cells comprise exposing the plurality ofB-cells to Epstein-Barr Virus (EBV) supernatant to produce a stimulatedplurality of B cells.
 26. The recombinant human monoclonal IFN-αantibody or IFN-α binding fragment thereof of claim 25, wherein theconditions allowing only a definite life span of the B cells compriseexposing the plurality of stimulated B-cells to CpG to produce anactivated plurality of B cells.
 27. The recombinant human monoclonalIFN-α antibody or IFN-α binding fragment thereof of claim 26, whereinthe conditions allowing only a definite life span of the B cells furthercomprise culturing the plurality of stimulated B-cells under oligoclonalconditions with an irradiated feeder cell population from a heathydonor.
 28. The recombinant human monoclonal IFN-α antibody or IFN-αbinding fragment thereof of claim 27, wherein the irradiated feeder cellpopulation comprises peripheral blood mononuclear cells (PMBCs).
 29. Therecombinant human monoclonal IFN-α antibody or IFN-α binding fragmentthereof of claim 24, wherein a framework region of a VH sequenceencoding the recombinant antibody comprises a sequence variation whencompared to a VH sequence of an antibody isolated from human serum. 30.The recombinant human monoclonal IFN-α antibody or IFN-α bindingfragment thereof of claim 29, wherein a framework region of a VLsequence encoding the recombinant antibody comprises a sequencevariation when compared to a VL sequence of an antibody isolated fromhuman serum.